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A D V A N C E S I N
I m m u
n o l o g y
V O L U M E 1 2
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CONTRIBUTORS TO THIS
VOLUME
NABIH
I.
ABDOU
KEITH
J. DORRINGTON
PETER
DUKOR
H . HEMMINCSEN
B .
D.
KAHAN
RICHARDM .
KRAUSE
HENRY
METZCER
J.
RADOVICH
R.
A.
REISFELD
MAXWELLRICHTER
D. W . TALMACE
CHARLESANFORD
GERALD
WEISSMANN
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ADVANCES
I N
Immunology
E D I T E D
B Y
F. J . DIXON, JR .
H E N R Y 6 . K U N K E L
Divis ion o f Experimental Pathology
Scrippr Clinic and Research Foundation
l a lollo, Cal i fornia
The Rockefel ler University
New York , New York
V O L U M E 1 2
1 9 7 0
ACADEMIC PRESS New York and London
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COPYRIGHT 1970, BY ACADEMIC PRESS, NC.
ALL RIGHTS RESERVED
NO PART
O F
THIS BOOK MAY BE REPRODUCED IN ANY FORM,
BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY
OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM
THE PUBLISHERS.
ACADEMIC
PRESS,
INC.
111 Fifth
Avenue, New
York,
New
York
10003
United Kingdom
Edition
published
by
ACADEMIC
PRESS,
INC. (LONDON)
LTD.
Berkeley Square House,
London WlXGBA
LIBRARY
F
CONGRESSATALOGARD
UMBER:
61-17057
PRINTED IN T HE UNITED STATES O F AMERICA
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CONTENTS
LIST OF CONTRIBUTORS
. .
. .
. .
.
.
. .
. vii
PREFACE . . . . . . . . . . . . . . . ix
CONTENTSF PREVIOUS OLUMES
. . . . . . .
. . xiii
The Sea rch fo r An t i bod ies w i th Mo lecu la r U n i f o rm i t y
RICHARDM. KRAUSE
I.
htroduction
.
.
.
.
.
.
.
. .
.
. .
.
1
11. Immunoglobulin Heterogeneity and Antibody Properties Indicative of
Limited Heterogeneity . . . . . . . . .
.
. 3
111. Human Antibodies with Restricted Heterogeneity .
.
. . .
10
IV. Experimental Generation of Antibodies with Restricted Heterogeneity
.
12
V. Myeloma Protein and Paraproteins with Antibody Activity . . . 43
VI. Discussion and Summation .
. . . . .
.
. . .
48
References .
.
. . .
.
.
.
. . .
.
.
53
S tr u ct u re a n d F un c ti o n o f y M M a c r o g l o b u l i n s
HENRYMETZCER
I.
Introduction .
. . . .
.
.
.
. .
11. Isolation and Storage of Macroglobulins . . .
.
111. Structure of Mammalian Macroglobulins .
.
. .
IV.
Subunits, Polypeptide Chains, and Proteolytic Fragments
.
VI. Functional Properties of Macroglobulins . . . .
VII. Genetic Basis
of
Macroglobulin Structure
. . . .
VIII. Biosynthesis and Metabolism of Macroglobulins .
.
IX.
Macroglobulin-Like Proteins from Nonmammalian Species
X. Role of Macroglobulins in the Immune Response . .
XI. Prospects .
. .
. . . . . .
.
References . . . . . . . . . .
V. Low Molecular Weight hlacroglobulin-Like Proteins
. . .
57
. . .
59
. . .
80
. . .
73
. . .
88
. . . 89
. . .
98
.
. .
100
.
. .
102
.
.
.
104
. . . 106
.
.
. 108
Transp lan ta t i on An t i gens
R.
A.
REISFELD
ND
B.
D.
KAHAN
I.
Introduction .
. . . .
.
.
. .
. .
.
.
117
11.
Extraction and Solubilization
of
Transplantation Antigens
.
.
.
119
111.
Physical and Chemical Nature of Transplantation Antigens . . . 145
IV. Biological Activity of Extracted Transplantation Antigens . . . 157
V.
Perspectives
. .
. .
.
. . . . . .
.
. 189
VI. Summary . . . . . . .
.
. . . . . 190
References
. . .
. .
. .
. . . . . . 191
V
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vi
CONTENTS
The Role
of
Bone Marrow in the Immune Response
NABIH
1
ABDOUAND MAXWELLICHTER
I
.
Introduction
. . . . . . . . . . . . .
I1. A Brief Survey of
the
Techniques Used for the Detection of Immuno-
competent Cells . . . . . . . . . . . .
I11. Bone Marrow as a Source of Immunocompetent Cells
. . . .
IV. Cells Involved in the Humoral Immune Response
. . . . .
V
.
Cell Interactions Resulting in the Induction of t he Im mun e Response
.
V l
.
Effects of Irrad iation on the Im m un e Response
. . . . . .
. . . . . .
. . . . . .
IX
. Bone Marrow Transplantation-Application . . . . . .
X
.
Conclusions
. . . . . . . . . . . . .
References . . . . . . . . . . . . .
VII
.
Cells Involved in Cell-Mediated Immunity
VIII. Cells Affected in Immunological Tolerance
Cell Interaction in Antibody Synthesis
D
.
W
.
TALMACE.. RADOVICH.
ND
H .HEMMINGSEN
I. Introduction
. . . . . . . . . . . . .
I1 Tw o Universes of Im mun ocom petent Cells
V. Enhancing Effect of Multiple Antigenic Determinants
. . . . . .
I11
.
The Adherent Cell
. . . . . . . . . . .
IV. Antigenic Competition . . . . . . . . . . .
VI
.
Enhancing and Suppressive Effects
of
Passively Administered Antibody
.
. . . . . . . . .
References . . . . . . . . . . . . .
. . . .
VII
.
Discussion and Speculations
The Role
of
Lysosomes in Immune Responses
GERALDWEISSMANN AND PETER DuKon
I . Introduction . . . . . . . . . . . . .
I1 Processing of Antigen by the Vacuolar System
. . . . .
IV
.
Lysosomes in Four Types of Immune Injury
. . .
. .
References . . . . . . . . . . . . .
. . . . . .
111. Mediators of Tissue Injury Found in Lysosomes
Molecular Size and Conformation of Immunoglobulins
KEITH J
.
DORRINGTON A N D CHARLES TANFORD
I. Introduction . . . . . . . . . . . . .
I1. Molecular Size of Immunoglobulins and Subunits . . . . .
I11. Conformation of Immunoglobulins and Subunits
IV. Recovery of Native Conformation Following Chain Dissociation and
Unfolding . . . . . . . . . . . . .
V
.
Conclusions
. . . . . . . . . . . . .
References . . . . . . . . . . . . .
AUTHOR
INDEX
. . . . . . . . . . . . . .
SUBJECT NDEX
. . . . . . . . .
. . . . .
202
203
207
213
237
244
246
251
255
257
258
271
272
273
276
276
277
277
279
283
285
304
306
322
333
334
341
366
375
376
383
409
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LIST
OF
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
NABIH
I. ABDOU,*The Harry Webster Thorp Laboratories, Division of
lmmunochemistry and Allergy, McGill University Clinic, Royal
Victoria Hospital, M ontreal, Q ue bec , Canada ( 201)
KEITH J. DORRINGTON,
.
R .
C .
Molecular Pharmacology U nit , Med ical
School, University
of
Cambridge, Cambridge, England
(
333)
PETER
DUKOR,
epartment of Medicine (C el l Biology and G ene tics ) ,
N e w York Unive rsity School of Medicine, Ne w York, N e w York (283)
H .
HEMMINGSEN,
epartment of Microbiology, University
of
Colorado
Medical C enter, Denver, C olorado
(271)
B. D. KAHAN,Department of Surgery, Massachusetts Gen eral Hospital,
Boston, Massachusetts ( 117)
RICHARDM.
KRAUSE,
Rockefeller University, New
York,
N e w
York
( 1 )
HENRYMETZGER,rthritis and Rh eu ma tism Branch, National Institute of
Arthritis and Metabolic Diseases, National lnstitute of Health,
Bethesdu, Maryland ( 5 7 )
J. RAWVICH,Department
of
Microbiology, University of Colorado M edi-
cal Center, Denver, Colorado (271 )
R. A. REISFEW,Laboratory
of
Immunology, National Institute
of
Allergy
and Infectious Diseases, Bethesda, MaryIand
(117)
MAXWELL
ICHTER,~
h e Harry W eb st er Th orp Laboratories, Division of
Immunochemistry and Allergy, McGill University Clinic, Royal
Victoria Hospital, Montreal, Q ueb ec, Canada (
201)
D.
W.
TALMAGE,
epartment of Microbiology, University of Colorado
Medical Ce nter, Denver, Colorado (271)
CHARLESANFORD,Department of Biochemistry, Du ke University M edi -
cal Cente r, Du rha m, North Carolina (333)
GERALDWEISSMANN,Department of Medicine (C ell Biology and G e-
net ics ) ,
New
York
University School of Me dicine, N e w
Yark,
N e w
York
(283)
Present address: Division of Immunology and Allergy, School
of
Medicine, Uni-
versity of Pennsylvania, Philadelphia, Pennsylvania.
Medical Research Associate, Medical Research Council, Canada.
vii
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PREFACE
T he pac e of immunologic research has quickened as the problems an d
potentialities of immunology have appealed to investigators with widely
differing backgrounds. The important subjects reviewed in the twelfth
volume of this serial publication represent the contributions of chemists
and biologists as well as immunologists. With the increasing scope of
immunology, thoughtful, authoritative summations of knowledge in
rapidly developing areas have become essential if those working in this
field are to stay familiar with its overall progress. W e are ind ebted to th e
authors of this volume for making this possible by taking the time to
share with us their expertise.
T he first article deals with t h e recently observed molecular uniformity
of antibodies to bacterial carbohydrate antigens. Dr. Krause, who has
initiated and carried out much of this research, discusses the practical
aspects of the production of uniform antibodies and points out their
potential usefulness. These molecules will play an important role in the
study of the amino acid composition and topography
of
the antibody
com bining site, an d by virtue of th eir allotypic homoge neity also should
aid in the definition of t he location an d chara cter of the various molecular
determinants of allotypic specificity. Early sequence data from uniform
rabbit antibodies already indicate significant homology in the variable
regions of the hu m an kap pa an d rab bit light chains, suggesting a common
ancestral relationship. Finally, because of their ready detectability by
physical means, uniform antibodies provide an additional means of
analysis of antibody responses an d the cellular an d / or genetic events
involved.
In the second chapter Dr. Metzger discusses in depth the physical,
chemical, and biological aspects of yM antibodies, a subject to which he
has con tributed m uch. T he physical characteristics, chemical composition,
and subunit structure of the typical circular yM pentomers are presented,
and the relationship of these molecules to low molecular weight yM-like
proteins is considered, The characteristics of the interaction
of
yM anti-
bodies with antigens, i.e., the nature and number of antigen combining
sites, and th e interaction of yM antibodies with the com pleme nt system
are compared to the corresponding properties of yG antibodies. Finally,
the biosynthesis and metabolism of yM antibodies and their peculiar
role in the immune response are considered.
Perhaps the major barrier to rapid achieverncnt in homotransplanta-
ix
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X
PREFACE
tion today is the paucity of our knowledge of the histocompatibility
antigens. These antigens, until recently, have resisted attempts at isola-
tion and concentration
so
tha t little could be lear ne d of their structure or
metabolism. In the third chapter, Drs. Reisfeld and Kahan, who are
responsible for much of the recent progress in this field, provide a critical
evaluation of the various procedures currently employed to extract these
antigens a nd to characterize the m chemically a n d physically. Th e various
biological activities of the histocompatibility antigens and their use in
assay systems for antigen and antibody detection and quantitation are
also described.
One of the most active areas of immunologic research involves the
participation and interactions of various cell types in the immune
response. The recognition of the requirement for cell interaction in the
antibody response has greatly complicated the problem of interpretation
of mu ch ex perimen tal data. T he next two chapters in this volume com ple-
ment each other admirably in presenting the current status of this
rapidly developing subject. Drs. Abdou and Richter give a detailed
account of the eviden ce supporting th e roles of multiple cell types in th e
immune response. In addition to defining what is known of the origins
and functions of macrophages, antigen reactive cells, and antibody form-
ing cells, they indicate apparent species differences in the sources and
roles of these cell types. They also point out those critical areas in which
evidence
is
still needed in order to allow the formulation of a reasonably
complete scheme for an immune response. Drs. Talmage, Radovich, and
Hemmingsen deaI with the same subject as it relates to our basic concepts
of the immune response. Their discussion centers on the kind of inter-
action between the two different universes of immunocompetent cells
and the probable nature of the immunologic specificity and function of
each.
Recent investigations into the nature and functions
of
lysosomes
indicate that these intracellular organelles may have the first and last
words in many immunologic encounters. In the sixth chapter, Drs.
Weissmann and Dukor present an authoritative summary of current
information relating lysosomes both to the initial processing
of
antigens,
a responsibility chiefly of macrophages, and to the phlogogenic events in
immunologic injury, a responsibility chiefly of granulocytes. The niacro-
phages via their lysosomal digestive capabilities appear to he not only
the initial regulators of the magnitude of an antigenic stimulus by de-
grading much potentially immunogenic material but also the processors
and perhaps conveyors of the final effective immunogen. The tissue
injury which develops after the interaction of antigen, antibody, and
complement or of antigen with sensitized cells is dependent to a large
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PREFACE
xi
extent upo n t he hydrolytic degradation
of
extracellular and intracellular
macromolecules by the lysosomal enzymes.
Clarifying the relationship between the biological properties of
immunoglobulins and their organization at the several levels
of
protein
struc tu re is one of the most exciting problem s in molecular biology tod ay ,
In the last chapter, Drs. Dorrington and Tanford discuss this relationship
as it concerns the higher levels
of
organization-size, shap e, an d internal
folding-of imm unog lobulin mo lecules. W hile availab le measurem ents
cannot yet define with certainty the three-dimensional configuration of
immunoglobulins, they do suggest reasonable working models which fit
antigen binding characteristics, electron microscopic appzarances, and
physical-chemical properties of these molecules.
As always, it is a pleasure to acknowledge the cooperation and
assistance of the publishers, who have done much to ensure the quality
of this series of volumes.
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Contents
o f Previous
Volumes
Volume
1
Transplantation Immunity and Tolerance
M . H A ~ E K ,. LENGEROV~,N D T. HRABA
Immunological Tolerance o f Nonliving Antigens
&CHARD T. MITH
Functions of the Complement System
ABRAHAM . OSLER
ABRAMB. STAVITSKY
J. H. HALE
WILLIAM0. WEICLE
P.
G.
H.
GELL
AND
B.
BENACERRAF
P. A. GORER
In Vifro Studies of the Antibody Response
Duration of Immunity in Virus Diseases
Fate and Bio logical Action of Antigen-Antibody Complexes
Delayed Hypersensitivity to Simple Protein Antigens
The Antigenic Structure of Tumors
AUTHOR
INDEX-SUBJECTNDEX
Volume 2
Immunologic Specificity and Molecular Structure
Heterogeneity of y-Globulins
The Immunological Significance of the Thymus
Cellular Genetics
of
Immune Responses
FRED
AFWSH
JOHN L.
FAHEY
J.
F.
A.
P.
MILLER, . H . E. MARSHALL,ND R. G. WHITE
G . J. V . NOSSAL
CHARLES . COCHRANEND FRANK .
DXXON
DERRICKOWLEY
Antibody Production by Transferred Cells
Phagocytosis
xiii
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xiv CONTENTS
OF
PREVIOUS VOLUMES
Antigen-Antibody Reactions in Helminth Infections
E. J. L. SOULSBY
Embryological Development of Antig-.
REED
A.
FLICKINGER
AUTHOR
INDEX-SUBE W INDEX
Volume
3
In Vifro Studies of the Mechanism of Anaphylaxis
K.
FRANK USTEN
AND JOHN . HUMPHFIEY
The Role of Humoral Antibody in the Homograft Reaction
CHANDLER. STETSON
Immune Adherence
D.
S. NELSON
Reaginic Antibodies
D. R. STANWORTH
Nature o f Retained Antigen and Its Role in Immune Mechanisms
DANH. CAMPBELL
ND
JUSTINE
S.
GARVEY
Blood Groups in Animals Other Than Man
W. H. STONE
ND M.
R.
IRWIN
Heterophile Antigens and Their Significance in the
Host-Parasite Relationship
AUTHOR
INDEX-SUB
ECT INDEX
C. R. JENKIN
Volume 4
Ontogeny and Phylogeny of Adoptive Immunity
Cellular Reactions in Infection
ROBERT. GOODND BEN W. PAPERMASTER
EMANUELUTERAND HANSRUEDYAMSEIER
JOSEPH
.
FELDMAN
MACLYNMCCARTYND STEPHEN. MORSE
Structure and Biological Activity of Immunoglobulins
SYDNEYOHENND RODNEY. PORTER
Ultrastructure of Immunologic Processes
Cell Wal l Antigens
of
Gram-Positive Bacteria
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CONTENTS
OF
1’REVIOUS
VOLUMES
xv
Autoant ibodies and Disease
H.
G. KUNKEL
AND E. M.
AN
J.
MUNOZ
Effect
of
Bacter ia a n d B acter ia l Products on A nt ib od y Response
AUTHORINDEX-SUBECT INDEX
Volume 5
Na tu ra l Ant ibod ies an d the Immune Response
STEPHEN V. BOYDEN
Imm uno logic al Studies wi th Synthet ic Polypept ides
Exper imental Al lergic Encephalomyel i t is and Autoimmune Disease
The Immunology
of
Insul in
Tissue-Specific Antigens
AUTHORINDEX-SUB
ECT
INDEX
MI-= SELA
F’HILIP
Y.
PATERSON
C. .
POPE
D. C. DUMONDE
Volume
6
Exper imental Glomerulonephr i t is: Imm uno logic al Events
and Pathogenetic Mechanisms
EMIL
.
UNANUE
ND FRANK.
DIXON
Chemical Suppression of Adapt ive Immuni ty
ANN
E.
GABRIELSON
ND
ROBERT
.
GOOD
Nucleic Acids as Ant igens
OTTO
J.
FLESCIAND
WERNER RAUN
In
Vitro Studies of Imm unolog ical Responses of l ym ph o id Ce lls
RICHARDW .
D ~ O N
J A R ~ S L A V
STERZLND ARTHUR . SILVERSTEIN
PHILIP
G.
H.
GELL
AND
ANDREW
.
KELUS
P. J. LACHMANN
Developmental Aspects
of
Immunity
Ant i -ant ibod ies
Cong
I
i n n a n d
I
mmu nocon g u in ns
AUTHOR
INDEX-SUB
ECT
INDEX
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xvi CONTENTS OF
PREVIOUS
VOLUMES
Volume 7
Structure and Biological Properties of Immunoglobulins
SYDNEYOHEN
ND
CESAR
UTEIN
Genetics of Immunoglobulins in the Mouse
MICHAEL,OTTER ND ROSELIEBERMAN
Mimetic Relationships between Group
A
Streptococci
and Mammalian Tissues
JOHNB. ZABRISKIE
DARCY
.
WILSON
ND
R.
E.
BILLINGHAM
JOHN P. MERRILL
lymphocytes and Transplantation Immunity
Human Tissue Transplantation
AUTHOR
NDEX-SUBECT INDEX
Volume
8
Chemistry and Reaction Mechanisms of Complement
HANS. MULLER-EBERHARD
Regulatory Effect of Antibody on the Immune Response
JONATHAN
.
Urn
AND
GORAN
MOLLER
The Mechanism of Immunological Paralysis
D.
W.
DRESSERND N. A. MITCHISON
In V i f r o Studies
of
Human Reaginic Allergy
ABRAHAMG.OSLER, AWRENCE. LICHTENSTEIN,N D
DAVID
. LEVY
AUTHOR
INDEX-SUB
ECT INDEX
Volume
9
Secretory Immunoglobulins
Immunologic Tissue Injury Mediated by Neutrophilic leukocytes
The Structure and Function of Monocytes and Macrophages
THOMAS. TOMASI,R.
AND
JOHNBIENENSTOCK
CHARLES. COCHRANE
ZANVILA.
COHN
J.
B.
HOWIEAND
B.
J. HELYER
The Immunology and Pathology
of
NZB Mice
AUTHOR
INDEX-SUBECT INDEX
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CONTENTS
OF
PREVIOUS
VOLUMES
Volume
10
Cell Selection by Antigen in the Immune Response
Phylogeny of Immunoglobulins
Slow Reacting Substance ofAnaphylaxis
Some Relationships among Hemostasis, Fibrinolytic Phenomena,
Immunity, and the Inflammatory Response
GREGORY. SISKIND
ND
BARU BENACERRAF
HOWARD
.
GREY
ROBERT . ORANGE
ND K.
FRANK
USTEN
OSCAR
D.
RATNOFF
KARL
HABEL
D. BERNARD
MOS
Antigens of Virus-Induced Tumors
Genetic and Antigenetic Aspects of Human Histocompatibility Systems
AUTHOR
INDEX-SUBECT INDEX
xvii
Volume 11
Electron Microscopy
of
the Immunoglobulins
N. MICHAELGREEN
HUGH
0.
MCDEVITT
ND
BARUJ BENACERRAF
The Lesions in Cell Membranes Caused by Complement
JOHN H. HUMPHREYND ROBERTR. DOURMASHKIN
Cytotoxic Effects of lymphoid Cells in Vifro
PETER ERLMANN
ND
GORANHOLM
H. S. LAWRENCE
IVOR
. BROWN
Genetic Control of Specific Immune Responses
Transfer Factor
Immunological Aspects
of
Malaria Infection
AUTHOR
INDEX-SUBECT INDEX
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The Search for Antibodies with Molecular Uniformity'
RICHARD M. KRAUSE
The R o cke f e l l e r U n i ve r s i t y , N e w Y o r k , N e w Y o r k
I. Introduction . . . . . . . . . . . . .
Immunoglobulin Heterogeneity and Antibody Properties Indicative of
Limited Heterogeneity
. . . . . . . . . . .
A.
Inimunoglohulin CIasses, SubcIasses, and Light-Chain Types .
.
B. Immunoglobrilin Charge Heterogeneity . . . . . .
D.
Immunoglobulin Allotypes
. . . . . . . . .
11.
C .
Heterogeneity of Antibody-Combining Sites . . . . .
E. Immunoglobulin Individual Antigenic Specificity . . . .
F.
Irnmunoglohulin Amino Acid Sequence . . . . . .
A. Rabbit Antihodies to Streptococcal Carbohydrates
. . . .
111. Human Antibodies with Restricted Heterogeneity
. . . . .
IV. Experimental Generation of Antibodies with Restricted Heterogeneity .
B. Rabbit Antihodies to Pneumococcal Capsular Polysaccharides . .
C. Isolation from Antisera of Antibodies to Bacterial Carbohydrates
.
D. Factors Influencing the Occurrence of High Antibody Responses
with Restricted Heterogeneity . . . . . . . .
E. Antibodies to Myoglobin and Angiotensin
. . . . . .
F.
Antibodies to Synthetic Antigens
. . . . . . . .
VI. Discussion and Summation
. . . . . . . . . .
References . . . . . . . . . . . . .
V. Myeloma Proteins and Paraproteins with Antibody Activity . . .
1
3
3
4
5
6
7
8
10
12
12
29
30
37
40
41
43
48
53
I. In t roduc t ion
W ithin the las t decade thcre has been
a
remarkable advance in the
knowledge
on
the chemical st ructure
of
thc: immunoglobulins. The
achievements are all the more remarkable because of the magnitude
of
immunoglobulin heterogeneity. Nevertheless, the immunoglobulins had
sufficient structural features in common
so
t ha t
R. R.
Porter
(1959),
Fleischman et al. ( 1963), Edelman and Cal l (1969), Edelm an an d Poul ik
(1961), and others w ere ab le to determ ine the four-chain st ructure, con-
sisting
of
one pair of light chains, an d
one
of heavy chains. Because all
of
the molecules share this basic structure, heterogeneity
is
a function of var-
iation in amino acid sequence. It was for this reason that a direct at tack
on the am ino acid sequence of the two antigenic binding sites has been
delayed, even though it was known that each
of the
tw o sites of IgG w as
localized to the variable p ortion of thc m o l c d e .
of Allergy and
Infectious Diseases, Crant N o . A1 08429,
m c l
I y a Clant-in-Aid from the American
Heart Association.
1
' Srtpportetl in
p i - t by
a
grant from the National Institute
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2 RICHARD
M.
KRAUSE
Once it was clear that a myeloma protein possessed molecular homo-
geneity and represented a single species of immunoglobulins, the way
was open to employ th e myeloma proteins to examine in muc h greater
de-
tail th e chemical struct ure of th e imm unoglobulins. Inde ed , the amino acid
sequence of one myeloma protein is now completely elucidated (Edel-
man and Gall, 19699, and there is much information on vast stretches
of many others. Despite such success, in all of this there is a nagging
reservation. Is a myeloma protein, the product of malignant cells, a
genuine representative of an antibody? Th e question has bee n answered
only in part, even thou gh a limited numb er of mouse an d hu m an myeloma
proteins possess reactivity for certain well-defined chemical antigens.
As
long as ambiguity surrounds this fundamental nature of the myeloma
proteins, any exploration of the topography of the antigen binding site
and a scrutiny of the structure-function relationships between antigens
and antibodies will require homogeneous antibody populations. It is for
this an d oth er reasons th at immunologists h ave sought specific antibodies
with molecular uniformity. It has been their goal to procure at will, and
in a reproducible and predictable fashion, homogeneous antibodies to
specific and defined antigenic determinants. But, the prospects for
achieving such a goal, until a few years ago, appeared bleak.
A number of excellent reviews have recently been published on the
subject of heterogeneity
(
Fleischman, 1966; Haber,
1968;
Franklin and
Frangione, 19 69 ). I n this series of volumes alone, at least four previous
reviews have described the s tru ctu ral heterogeneity of the immuno-
globulins and the molecular and functional heterogeneity of specific
antibodies (Fa he y, 1962; Cohen an d Porter, 1964; Cohen and Milstein,
1967; Grey, 1%9).It is not the purpose of this review to recapitulate the
evidence for molecular heterogeneity of the immunoglobulins as well
as most specific antibody preparations.
T he review is th e first of this series to deal exclusively with th e search
for antibodies with molecular uniformity. It will
be
confined to the
history of this quest, an d will not review in d eta il th e chemical structure
of the immunoglobulins or the evidence for molecular and functional
heterogeneity of immunoglobulins and specific antibodies to a wide
range of different antigens. The references cited will be selective and
representative rather than exhaustive, and the final selection will un-
doubted ly represent t he un intentional bias of th e author. T h e focus will
remain centered o n the o ccurrence of antibodies with uniform properties
following specific antigenic stimulation. Human and mouse myeloma
proteins or paraproteins with antibody activity will be included because
use
of
thcse proteins remains an alternative approach to procure anti-
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SEARCH FOR ANTIBODIES
WITH
MOLECULAR
UNIFORMITY
3
bodies with molecular uniformity. No attempt will
be
made he re to
include a ll aspects of this work.
The review has been divided into several sections. First
a
brief sum-
mary is presented of the hallmarks of immunoglobulin heterogeneity
and the criteria that must be used to judge the restricted heterogeneity
or
molecular uniformity of an antibody preparation are considered. Then
human antibodies to certain selected antigens which appear to possess
less heterogeneity than normal y-globulin are discussed. A brief account
of these human antibodies is important because their occurrence has
sparked effo rt to procure antibodies w ith m olecular uniformity in ex-
perimental animals. Next the experimental approaches to generate anti-
bodies w ith limited heterogeneity in animals a re described. Em phasis
is placed on the evidence for the molecular uniformity of rabbit anti-
bodies to bacterial carbohydrate antigens. The section after that deals
with human and mouse myeloma proteins and paraproteins which have
reactivity for specific antigenic determinants. The Discussion and Sum-
mation section comments about the potential usefulness
of
homogeneous
antibodies for examining the structure-function relationships of antigens
an d antibodies an d for p robin g the genetic machinery which is responsi-
ble for antibody diversity.
II. Im m u nog l obu l i n H e t e r ogene i t y an d A n t i b od y P r ope rt i es
Ind i ca t i ve o f L im i ted He te rogene i t y
I t is self-evident t h at a specific antibody
is
not a homogcneous protein
unless it lacks all of the manifestations of immunoglobulin heterogeneity.
These manifestations include: isotypic variation, charge heterogeneity,
functional heterogeneity, and allotypic variability. Specific antibodies
tha t are generated in response to most antigenic stimuli ar e heterogeneous
as judged by all of these manifestations or by any combination of them.
Individual antigenic specificity or idiotypy refers to the antigenic in-
dividuality
of
a myeloma protein or a uniform antibody population.
Heterogeneous immunoglobulins lack this special characteristic.
A.
IMMUNOGLOBULINLASSES,UBCLASSES,ND
Five major classes of immunoglobulins have been recognized in the
human
(IgG,
IgA, IgM, IgD , an d I g E ) , and their dis tinguishing fea-
tures have been recently reviewed (Fran klin and Frangione,
1969;
Grey,
1969).
Subclasses are described for IgG, IgA, and IgM. Each of these
classes and subclasses possess either K o r h light chains, but the dis-
tinguishing characteristic of the class or subclass is the chemical structure
LIGHT-CHAIN YPES
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4
RICHARD
M.
KRAUSE
of the heavy chain. These characteristic heavy chains have been desig-
nated y,
01,
p,
6, and
c
for IgG, IgA, IgM, IgD, and IgE, respectively.
It is obvious that an antibody is not uniform or homogeneous unless
it belongs to only one class and subclass of immunoglobulin and that
its light chains are either
K
or A. In some cases, it is possible to isolate
a
specific antibody from an antiserum that consists of only one class. But,
an antibody preparation, although all of one class, is commonly a hetero-
geneous mixture as judged by other criteria.
B. IMMUNOGLOBULINHARGE ETEROGENEITY
The disperse distribution of the 7-globulins by electrophoresis is an
indication
of
charge heterogeneity. All the classes exhibit this hetero-
geneity. In contrast, each myeloma protein possesses a marked uniform-
ity of charge which results in an extremely restricted electrophoretic
mobility. Most purified antibodies exhibit charge heterogeneity, although
some preferential selection has been observed by Nussenzweig and Bena-
cerraf (1967) for certain antibodies in guinea pigs and by Sela (1967)
for rabbit antibodies to synthetic polypeptides. In neither case, however,
was this restriction in charge as narrow as that seed for the myeloma
proteins, and in both instances the antibodies were heterogeneous by
other criteria. Disc electrophoresis in acrylamide gel of reduced and
alkylated immunoglobulins and of most of the specific antibody prepara-
tions reveals a number of distinct fractions for both the light and the
heavy chains ( Reisfeld and Small, 1966). These individual fractions
have been isolated, and analysis revealed differences in amino acid com-
position
(
Reisfield,
1967).
Charge heterogeneity of the immunoglobulins facilitates their separa-
tion into fractions by chromatography. Feinstein ( 1964) separated
rabbit IgG into four chromatographic fractions; each successive frac-
tion from the column had an electrophoretic mobility greater than the
previous one. The light- and heavy-chain mobilities for the four chroma-
tographic IgG fractions were also compared. Each possessed heavy chains
with identical mobility, but the Iight-chain mobility was parallel to that
of the intact IgC chromatographic fraction from which it was derived.
Studies, to be described later, for specific rabbit antibodies are largely in
agreement with these findings.
Taken altogether, these studies indicate that the charge heterogeneity
of an antibody is an indication of molecular heterogeneity. I t is unlikely
that an antibody is uniform unless the light chains migrate as one major
component by disc electrophoresis. This certainly appears to be the case
for rabbit antibodies to streptococcal carbohydrates ( Eichmann et al.,
1970a). The one qualification of this generalization stems from the work
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SEARCH F O R ANTIBODIES
WITH
MOLECULAR UNIFORMITY 5
of Awdeh
et
al. (1 96 7 ). Bence-Jones p roteins isolated from cells gave
a single ban d on starch gel electrophoesis, whereas this protein incubated
in serum gave scveral bands. Presumably, some alteration of the protein
occurred in the serum which resulted in charge heterogeneity. Further-
more, charge heterogeneity of mouse myeloma light chains is related to
the sialic acid content (Melchers et
a?.,
1966) .
Although the light chains of a myclonia protein will commonly
migrate in one major band on polyacrylamide disc electrophoresis, the
pattern is more complex with respect to the heavy chains. The heavy
chains migrate in at least several bands (Dorner
et
al . , 1969). Such
heterogeneity may be a reflection of variability in carbohydrate content
and not due to heterogeneity in the amino acid sequence. The heavy-
chain pattern of the myeloma proteins is less complex than that of normal
y-globulin, however. Normal heavy chains migrate in at least ten bands.
C. HETEROGENEITYF ANTIBODY-COMBININGITES
Antibodies against simple antigens or haptens exhibit heterogeneity
in th e characteristics of th e com bining sites. Such heterogeneity can
be detected in two different ways. First, the antibodies vary in the
affinity for the antigen an d, second, they vary in th e size of the antigenic
determinant to which they bind. Affinity heterogeneity has been studied
in detai l for rabbit antibodies to 2,hdini trophe nyl ( D N P ) (Eisen, 1966 ).
When DNP groups
on
protein substances are employed as antigens, the
antibodies exhibit
a
wide range of binding energy to DNP. Furthermore,
there appears to be combining site heterogeneity as a function of time
after immunization (E isen and Siskind, 19 64) . Th e average binding con-
stants revealed a progressive increase from about lo5L / M for antibodies
recovered 2 weeks after
a
small immunizing dose to about 10' L / M for
antibodies recovered 8 weeks after this dose. This change in affinity
remains to be explained.
Variability in the bind ing of an tibody to haptene s of different sizes
has been shown for human antidextran and anti-blood-group substances.
For example, antidextran antibody was recovered from
a
Sephadex
column by sequentid elution with oligosaccharides of increasing length
(Schlossman an d Kabat, 1 96 2) . Precipitation of d extran by t h e anti-
body eluted with the larger hapten was inhibited by the larger oligo-
sacchardies, whereas di- and trisaccharides were less effective. Similar
studies have been reported recently for human antibodies to blood group
A
substance (Mo reno and Kabat , 19 69).
In
this case, the antibody was
recovered from an inimunoabsorbent column by elution with N-acetyl-
galactosamine and by a pentasaccharide. N-Acetylgalactosamine did not
inhibit precipitation between the blood group A substance a nd t he an t i-
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6 RICHARD M.
KRAUSE
body recovered with the pentasaccharide. On the other hand, N-acetyl-
galactosamine did inhibit the precipitation when the antibody employed
was that recovered by N-acetylgalactosamine elution. It is suggested
that the antibodies eluted by N-acetylgalactosamine possess a combining
site which is smaller in size than that of those antibodies eluted with the
pentasaccharide.
D.
IMMUNOGLOBULINLLOTYPES
Allotypic variation of the immunoglobulins is due to intraspecies anti-
genic differences which are inherited in a simple Mendelian manner
(Oudin, 1966). In certain cases, these antigenic differences have been
shown to be associated with variations in the amino acid sequences for
both the light and heavy chains and were first described by Oudin (1956)
in rabbit, by Grubb (1956) in man, and by Herzenberg (1964) in
mouse. The localization
of
these allotypic antigenic markers to certain
specific amino acid sequence regions of each chain has been a subject
of much interest because the outcome has ramifications in regard to
genetic theories of antibody formation, All of this and the associated con-
troversy have been reviewed elsewhere (Herzenberg, 1964; Hood et al.,
1967; Baglioni et al., 1968; Natvig et al.,
1968;
Koshland, 1968; Wilkin-
son, 1969; Prahl
et
al.,
1970; Hood and Talmadge, 1970; Vice
et
al.,
1969;
Dubiski, 1969).
The allotypic markers of rabbit IgG and the localization of each on
either the light or the heavy chain are identified in Fig.
1.
Such a dia-
gram has been constructed from the information in the references cited
above. As a general rule, specific antibodies recovered from heterozygous
rabbits possess both allotypes for each of the groups for which they are
heterozygous, although a modest to a marked selective shift in the pro-
portion of the markers has been observed (Catty
et d. ,
1969).
A1 lo types
FIG. 1. Schematic diagram of y-globulin and the approximate location of the
allotypic markers.
(
Modified after Kindt, 1970.)
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SEARCH
FOR
ANTIBODIES
WITH MOLECULAR UNIFORMITY
7
E. IMMUNOGLOBULINNDIVIDUAL
NTIGENIC PECIFICITY
Individual antigenic specificity was initially described
b y
Kunkel
(1965 ) a n d his colleagues as a property of th e myeloma proteins. Th is
property is a reflection of the antigenic individuality
of
the variable
region of th e protein a nd is, therefore, c o n h e d to th e Fa b portion. Be-
cause of this individuality, the myeloma protein induces an anti-antibody
in a heterologous species which after absorption with pooled human
Fraction I1 reacts specifically with the myeloma employed for immu niza-
t ion (Grey e t al., 19 65). One interpretation of these studies is that each
antigenically distinct myeloma protein is representative of a normal
y-globulin molecule an d tha t the total pool of norm al y-globulin in
the
serum is the summation of a very large number of different molecules
or species each bearing one
of
these unique antigenic specificities
(Pernis, 19 68 ). Although examination of m any hu m an paraproteins h as
revealed only occasional instances of cross-specificity (Williams et al.,
1Q68), most theoretical estimates suggest that normal serum contains
y-globulin of such antigenic diversity that the number of different
molecules, each with a distinctive individual antigenic specificity, exceeds
several thousand (Pernis, 1968; Kunkel, 1 97 0). Such a large nu mb er
is consistent with the possibility that there are between
1000
and
10,000
different heavy chains, each with distinctive features in the amino
acid sequences, and a n equally large num ber of light chains. T h e many
possible combinations of these light and heavy chains to form a complete
molecule is a very large n u d e r .
Parallel studies on antigenic specificity have also been done on a
selected small number of human antibodies which, by several critiera,
exhibit restricted heterogeneity (Kunkel et al., 1963). Such antigenic
individuality is also a manifestation of a specific antigenic determinant
associated with the variable region of t h e molecule.
Antigenic individuality of specific antibodies has been detected in
a somewhat different way by Oudin and Michel (1963) and by Kelus
and Gell
(1968; Gell and K elus, 196 7). In these studies, th e anti-
antibodies are raised in rabbits that have the same allotypy as the donor
rabbit. Such antigenic individuality has been termed “idiotypy.” Perhaps
th e most troublesome obstacle fo r an un amb iguous interprctatioii of these
studies on idiotypy is that clearly defined and isolated antigens and anti-
bodies have not been employed in many
of
these experiments (Oudin
a n d Michel, 19 69 a,b ). Recently, however, idiotypy has been described
by Daugharty et (11. (1969) for antibenzoate antibodies in rabbits. In
this case, the phenomcnon of idiotypy has been demonstrated with
isolated and well-defined antigens and antibodies. As was noted above,
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8
RICHARD
M.
KRAUSE
there has been a good deal of speculation on the biological significance
of individual antigenic specificity, and similar discussion surrounds
idiotypy. Setting aside these theoretical considerations, from the prac-
tical point of view, the demonstration that all of the molecules in an
antibody preparation possess the same individual antigenic specificity
or idiotypy is probably the single most useful criterion which is indica-
tive of molecular uniformity short of amino acid sequence analysis.
F. IMMUNOGLOBULIN
MINO ACID
SEQUENCE
Final, irrefutable proof for either heterogeneity or homogeneity of
immunoglobulins and specific antibodies is obtained from amino acid
sequence data. It must be admitted, however, that appreciable degrees
of heterogeneity may escape detection because the methods employed for
complete amino acid sequence analysis are not quantitative. Despite
such reservations, much has been learned about the degree of homo-
geneity of antibodies and myeloma proteins through an examination
of
either partial or complete sequences. The magnitude of the labor to
obtain a complete sequence of a homogeneous protein has spurred on
the employment of indirect approaches to determine the degree of
heterogeneity of an immunoglobulin or antibody preparation. I t is far
easier, for example, to determine the
K-
and A-chain composition by
serological means than by amino acid sequence of the C-terminal portion
of the light chains.
N-terminal amino acid analysis of the light chains of normal rabbit
IgG and human Bence-Jones proteins leaves no question about the
heterogeneity of the former and the homogeneity of the latter (Hood
et al.,
1989).Typical, quantitative, Edman analysis data are presented
in Table
I.
Tabulated here are the results of the quantitative Edman
analysis for the first three N-terminal residues of the light chains of
normal IgG recovered from a single rabbit and a human Bence-Jones
protein. The amount of each amino acid at each position has been de-
termined from a HC1 hydrolyzate of the PTH’” amino acids (Van Orden
and Carpenter,
1964).
The per cent yield has been calculated from the
nanomoles of the specific amino acid and the total nanomoles of all
the amino acids recovered at a particular sequence step. The per cent
recovery at each position has been calculated from the nanomoles of
all the amino acids at each sequence step and the nanomoles
of
the
dry weight protein used for the Edman procedure. It is obvious that
for each of the first three N-terminal positions, several major amino acid
alternatives exist for the nonimmune IgG
of
a single rabbit, whereas
one major amino acid is present for each position in the Bence-Jones
protein. Studies of a qualitative nature, but similar in design, by Doo-
In Phenylthiohydantoin.
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SEARCH
FOR ANTIBODIES WITH
hlOLECULAR
UNIFORMITY 9
TABLE
1
AMINO
ACID
RESIIKESAr
N-TERMINAL
E Q U E N C E POSITIONS
,
2,
A N D 3
FOR
THE
CONTROL
ENCE-JONES
ROTEIN
Hackney)a,*
LIGHT
HAINS
O F P R E IM M I i N E
Raoui’r
~ - G I , O H I T I , I N
N D
FOR
T H E
Position
1
Position 2 I’osition
3
Rerov- Recov- Recov-
Yield ery Yield ery Yield ery
Light chains ( ) 96) ( ) ( I
( ‘23)
( I )
Rabbit
R32-85
Ala 54
preimmiine
Ile 6
Bence-Jones protein Glu
1 0 0 33
(Hackney) aiialysis
1
Bence-Jones protein Glu
96
(Hackney) analysis 2 Gly
2
63
Asp
2
Asp
34 Val
28
Gly
1.5
Glu 5
Gly
6
Ile 67
Glu
6
a Quarititat>iveEdman procedure employed. The per cent yield
of
each amino acid
at each position has been calculated from the nanomoles of the specific amino acid and
the total nanomoles of all the amino acids recovered a t t.hat particular sequence step.
The per cent
recovery
a t each position has been calculat,ed from the nanomoles of
all
amino acid3 at each sequence step and the dry weight of protein used for the Edmari
procedure. Except for position 1, residue yields of 4 % or less are not listed.
*
From Hood et al.,
1969.
little (1965) have shown that there are just as many amino acid alterna-
tives for each of the first six N-terminal positions for specific isolated,
rabbit antibodies to DNP as for rabbit IgG. Such findings are an indica-
tion that specific antibodies can have
a
heterogeneity indistinguishable
from normal IgG. Similar evidence will be reviewed later which suggests
that the light chains of certain antibodies have a single amino acid
present at each of the first three N-terminal positions and in this respect
resemble the Bence-Jones proteins.
It is not within the scope of this review to explore in greater detail
the structural and functional heterogeneity of antibodies or to speculate
on the biological significance of heterogeneity. I t is conceivable,
of
course, that the excessive effort expended to achieve antibody hetero-
geneity is, in fact, an affidavit that life will be preserved. As Eisen
(1966)
has pointed out, diversity of ligand-binding sites within an anti-
body population assures a wide range of cross-reactivity for antibody
molecules of a particular specificity. Thus, the more heterogeneous the
immune response to viruses and bacteria, the more broadly based the
spectrum of acquired resistance.
A
heterogeneous immune response to
one set of virus antigens, for example, may broaden the acquired im-
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10
RICHARD
M. KRAUSE
munity against related viruses and against new viruses arising from
the natural unrelenting mutability of such microbes.
I l l
Human Antibodies with Restricted Heterogeneity
Cited in the li terature are numerous examples of specific antibodies
that exhibit much less heterogeneity than the total complement of t he
immuno globulins in th e antiserum from w hich th e antibodies are isolated.
This literature has been included in reviews on immunoglobulins by
Fleischman (1966) and Haber (1968) and such documentat ion need
not be repeated here.
Early in the 196O’s, studies on human antibodies to various carbo-
hydrates suggested that carefully selected human antibody populations
might
be
less heterogeneous than had been commonly supposed. In-
dividual antigenic specificity was demonstrated by Kunkel et al. (1963)
for antihuman blood group A, anithuman blood group B, antidextran,
antilevan, and antiteichoic acid antibodies isolated from human sera.
A
typical experiment is depicted in Fig. 2. The antibody to blood group A
reacted specifically with its absorbed anti-antiserum. No cross-reaction
occurred with four y-globulin preparations
of
different types or with
seven heterologous isolated anti-A antibodies. Subsequent studies re-
vealed that the light chains of some of these antibodies migrated in
one major band in 8
M
urea starch gel electrophoresis (Edelman and
Kabat, 1964).T he distribution
of
the 7-globulin genetic factors, Gm (a),
G m (
b) ,
and Inv ( a ) , was studies for the isolated antibodies against
FIG. 2.
Agar plate analysis showing the specific reaction
of
isolated anti-A
antibody, Th, with anti-Th antiserum (A). Peripheral wells 2-5 contain y-globulin
preparations
of
different types, and wells
6-12
contain heterologous isolated anti-A
antibodies. The antiserum
( A )
was absorbed with
5
mg. Fraction I1 per milliliter
of serum. (From Kunkel
et
al.,
1963.)
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SEARCH FOR AXTIBODIES WITH MOLECULAR UNIFORMLn
11
dextran, levan, teichoic acid, an d blood g ro up A substances (Allen d al.,
1964). Although the majority of these antibodies contained all of the
genetic factors determined in the donor’s whole y-globulin, in
a
number
of the antibodies these factors were present at very different concen-
trations. Most important, in a few instances, specific factors were not
detected despite their presence in the individual’s whole y-globulin. In
these cases the distribution of the genetic factors appeared to approach
the selective occurrence of these factors in mycloma proteins,
These studies on human antibodies were consistent with findings of
a similar nature reported somewhat earlier by Gel1 and Kelus (1962).
They observed the absence of one and possibly two allotypes in an
antihapten antibody isolated from
a
rabbit , and Rieder and Oudin
(1963) demonstrated alterations in the normal ratios of various allotypic
markers in isolated antibodies to bovine serum albumin and DNP-bovine
y-globulin. Taken together, such findings raised the hopeful question,
“Are specifically purified antibodies of an individual, like pathological
proteins, discernably less heterogeneous than his total 7-globulin?” (Edel-
man a nd Kabat , 1964).
If
such proves to b e the case, Edelman an d Kabat
went on to comment, “then the analogy of these antibodies to myeloma
proteins and Bence Jones proteins may have useful application in
de-
tailed chemical studies of antibodies” and, in particular, may prove of
value for the “purpose of determining the amino acid sequence of the
structure of their combining sites.” Such objectives have sustained the
search for the means to procure in
a
reproducible and predictable
fashion, antibodies with molecular uniformity.
Recent studies by Yount et al. (1968) indicate that the human anti-
levan referred to above (Allen e t aZ., 1964) is a remarkably uniform
antibody.
It
consists exclusively of 7G L heavy chains an d
K
light chains
and exhibits, as detected by quantitative assays, a selective absence of
genetic markers.
It is also clear from these studies of Yount
et
al.
(1968)
that an anti-
serum may possess several sets of antibodies against a single antigen. For
example, the degree of homogeneity of antibodies to dextran recovered
from the serum
of
one individual was progressively enhanced by subfrac-
tionation of the antibodies on the basis of antibody specificity for the
glycosidic linkage or combining site size rather than by physicochemical
methods. An isomaltohexose eluate of dextran y G 2 antibody from two
subjects
was
priinarily
K,
and isonialtotriosc eluate
was
primarily
A.
The impression which is gaincd from all of these studies
is
that
human antibodies to carbohydrates may possess, to
a
surprising degree,
properties which are iadicative of restricted heterogeneity. This raised
the possibility that antibodies to carbohydrates, produced in experi-
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12 RICHARD M. KRAUSE
mental animals, might have much less heterogeneity than the antibodies
to the commonly employed synthetic haptens. The use of rabbits was
prompted by the knowledge that they may respond with high levels of
antibodies to carbohydrate antigens following intravenous immunization
with bacterial vaccines, most notably streptococcal and pneumococcal
vaccines. A major consideration is the supply
of
antibody which can be
obtained from animals. If homogeneous antibodies are to be recovered
from experimental animals in sufficient yield for structural studies, im-
munization must lead to antibody concentrations which are similar to
the concentrations of the myeloma proteins in man.
IV.
Experimental Generation
of
Antibod ies with Restricted Heterogeneity
A. RABBITANTIBODIES
O
STREPTOCOCCALARBOHYDRATES
It is apparent from the discussion thus far that immunization with
any on e of sev eral bacterial antigens might provoke an tibodies with re -
stricted heterogeneity. The idiotypy of antibodies in the antisera of
rabbits immunized with salmonellae suggests, for example, that these
antibodies have less heterogeneity than the nonimmune y-globulin. Use
of the streptococci, however, to generate a restricted immune response
has several advantages over the salmonellae. First,
the
immunochemistry
of the streptococcal carbohydrate antigens is less complex than that of
salmonellae. Sceond, antisera of rabbits immunized with streptococcal
vaccines had been known to contain in some instances very high con-
centrations of antibody to the carbohydrate. Initial studies indicated that
in certain rabbits, antibodies to the carbohydrates had an electrophoretic
uniformity and a serum concentration in many ways as remarkable as
that of the myeloma proteins (Osterland et al . , 19 66). Recent evidence
from other laboratories suggests that this is also the case for rabbit anti-
bodies to Type
I11
and Type V III pneumococcal capsular polysaccharide
(H ab er , 1970; Pincus et
al.,
1970a,b) .
Considerable detailed information is now available on the rabbit im-
mune response to streptococcal carbohydrates, and a condensed review
of this work will be p resented h ere . The points to be emphasized include:
the method of immunization; the immunochemistry of the streptococcal
carbohydrate antigens; the immune response; and evidence for the
molecular uniformity
of
the antibody.
1 .
Method
of
~ m m u ~ i z ~ t i o n
A
good deal of experience, much of it empirical, indicates that the
highest levels of antibodies to the streptococcal carbohydrate are
achieved in the rabbits by intravenous immunization with a vaccine of
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14 RICHARD M. KRAUSE
hyaluronic acid capsule, but this is washed away during the preparation
of the vaccine. Pepsin removes the bulk of the surface protein antigens,
M,
T,
and R (Lancefield, 1962). What remains and what is injected in-
travenously is intact heat-killed streptococci with a superficial cell wall
surface of carbohydrate antigen which masks the underlying muco-
pep tide matrix (Krause an d McCarty, 19 61) . Less than 10
f
t he dry
weight of the vaccine consists of carbohydrate. The remainder consists
of all the other cellular elements which have not been eliminated during
vaccine preparation. Yet,
it
is a remarkable fact that as much as
95%
of the 7-globulin in immune antisera is antibody to the octermost carbo-
hydrate ant igen (Braun et al., 1969).
Rabbits are immunized intravenously 3 times a week for
4
weeks.
With each injection, 0.5 ml.
of
vaccine
is
given the first week; there-
after 1 ml. is injected. Maximum antibody levels are seen between the
fou rth and eighth d ay after the last injection, as determined by qua ntita-
tive precipitin tests with the purified Group
C
carbohydrates (Fleisch-
man et al., 1968; Braun et al., 19 69 ). Although excellent antibody levels
may be achieved with the primary immunization, many rabbits require
a second series of injections after an interval of 3 to 5 months. The
second series of injections is similar to th e first, except th at m aximum
antibody levels are usually achieved with only
3
weeks of injections.
2.
Immunocheinistry of Streptococcal Carbohydrate
The immunochemistry and chemistry of the carbohydrate of Group
A streptococci has been studied in details by McCarty and others, and
G r o u p A c a r b o h y d r a t e
M.W.- 10,000
38 m o l e s
r h a m n o s e
:
0
17
m o l e s
N - o c e t y l
g l u c o s o m i n e :
@
FIG.4.
Schematic diagram
of
the chemical structure
of
the
Croup
A carbo-
hydrate antigen extracted from Group
A
streptococci. (F rom Krause, 1970. )
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
15
1
this work has been reviewed elsewhere ( McCarty and Morse, 1964).
The picture which emerges for the structure of this carbohydrate is
depicted in Fig.
4.
Th e antigen extracted from th e cell wall by chemical
means is undo ubtedly heterogeneous with respect t o size, bu t, certainly, a
portion of the extracted material has a molecular weight in the range of
10,000.
A
mole of such antigen possesses
17
moles
of
N-acetylglucosamine
a n d 38 moles of rhamnose. Eleven of the 17 moles of N-actylglucosamine
can be selectively stripped off with
p-N-acetylglucosaminidase,
leaving a
residual polymer which
is
predominantly rhaninose. McCarty
(
1958 ) has
shown that th e terminal /3-N-acetylglucosamiiiide residues are the imm uno-
dom inant determinan ts of specificity. Com panion carbo hydrates to this one
of Group
A
have b een isolated from C ro up A-variant
(
McCarty, 1956)
and
Group C streptococci ( Krause and McCarty, 19 62). Each of these antigens
ar e isolated from ei the r th e whole bacteria or cell walls by m ethods de -
scribed by Krause and McCarty (1961) and modified by Krause (1967).
T he evidence suggests tha t a similar rhamnose polymer is shared by each,
but, in the case of the Group C carbohydrate, terminal N-acetylgalactos-
aminide residues confer antigenic specificity; whereas, in the case
of
t he
Group
A
antiserum
R22-85
40r
30
I
E
0 0.2 0.4 0.6
Group
C
antiserum
R24-35
/*-•
/(C-CHO
Group
C
antiserum
R24-35
/*-•
C - C H O
i *
-
/ *
A - C H O
0-o-o-na-n
1 1 1 1 1 1 1 1 1 1 1
A - C H O
0-o-o-na-n
1 1 1 1 1 1 1 1 1 1 1
0 0.2 0.4
0.6 0.8
1.0
rng. C H O / 1.0 ml.
serum
FIG.5 .
Quantitative precipitin reactions between Groups
A
and
C
streptococcal
carbohydrates and the
hoinologous
and heferologous group-specific antisera. (See
Braun et d. 969, for details
of
the method.)
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16 RICHARD M.
KRAUSE
80
Group A-variant, the antigenic specificity is dependent upon the rham-
nose moiety itself.
The
antigenic specificity of this rhamnose moiety for
both Groups
A
and C carbohydrates is masked by the terminal amino
sugar residues.
The specificity of the streptococcal antisera for the homologous
carbohydrates is illustrated by the quantitative precipitin data in Fig. 5.
Group A antibody gives no appreciable cross-reaction with Group
C
antigen and vice versa; Group C antibody gives no significant cross-
reaction with the Group A antigen (Krause and McCarty, 1962). Recent
evidence from Kabat and co-workers (1970) and from Greenblatt and
-
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SEARCH FOR ANTIBODIES WITH
MOLECULAR
UNIFORMITY
17
Krause ( 1970) indicate that the terminal N-acetylgaIactosaminide resi-
dues of the Group C carbohydratc arc &-linked to the subterminal rham -
nose-a fe at ur e which
probably
accounts for the remarkable lack
of
cross-reactivity between Groups
A
and
C
carbohydrates. If the terminal
N-acetyl amino sugars were linked in a similar fashion in both cases
(i.e., either both
(Y
or both
/I ,
t is probable that greater cross-reactivity
would
be
detected.
The streptococcal carbohydrate precipitin reactions are readily in-
hibited by the characteristic N-acetyl amino sugar. Inhibition tests,
depicted in Fig. 6 (Krause and McCarty, 1962) were carried out at
antigen-antibody equivalencc. Wit h these particuIar antisera, 50% nhibi-
tion was achieved with approxiinately
3
mg./ml. of N-acetylglucosamine
in the case of G rou p
A
precipitin reaction and no inhibition was observed
with N-acetylgalactosamine. Likewise, 3 mg./ml. of N-acetylgalactos-
amine inhibited Group C precipitin reaction and no inhibition was
achieved with N-acetylglucosamine. Such a striking inhibition of the
precipitin reaction with a monosaccliaride has served as the basis for
recovery of specific antibody from immunoabsorbents by elution with
N-acetyl amino sugars. This procedure will bc described in a later section.
3.
Evictence
for
Restricted Heterogeneity
of
Antibodies
to Streptococcal Carboliydrates
The initial observations of Osterland et aZ. (1966) suggesting that
antibodies to streptococcal carbohydrates may have a remarkable mo-
lecular uniformity arc noted in Figs. 7 and 8. In Fig. 7 are depicted
th e microzone electrophoretic patterns of seruni from a r ab bit prior to
immunization and an antiserum collected from the same rabbit after 4
weeks of im munization with G ro up A-variant vaccine.
A
sharp narrow
band is seen in the 7-globulin region. The protein in this band is pre-
dominantly antibody to the group-specific A-variant carbohydrate. Im-
FIG.7 . hlicrozone electrophoresis
of
a rabbit serum collected prior to immuniza-
tion
( top
frame) and
of an
antiseriini collected after completion
of 4
weeks of
intravenous immunization with Group A-variiunt streptococci. ( From Ostcrland
et ul . , 1966.)
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18
RICHARD M. KRAUSE
S e ru m 4
Day 22
Serum 8
Day 71
FIG.8.
Tracings of the densitometric scans of microzone electrophoretic patterns
on
sera of a rabbit collected prior (serum l), duiing (serum 4), ust after completion
(serum 5) , and 42 days after completion (serum 8 ) of 4 weeks of intravenous
immunization. Sera
1
and 5 were depicted in
Fig.
7. (From Osterland et
al.,
1966.)
munoelectrophoresis reveals that it is entirely IgG.
It
is a
7s
protein
as determined in the analytical ultracentrifuge.
The transient nonmalignant nature of this response is indicated
by
the immunoelectrophoretic patterns on serial sera collected well after
immunization.
As
is noted in
Fig. 8,
during the interval between day 29
and day 79, a t which time no vaccine was adm inistered, the antibody pe ak
receded and the 7-globulin concentration approached the preimmune
level.
Initial evidence that antibodies in an antiserum such
as
this possessed
restricted heterogeneity included the monodisperse electrophoretic dis-
tribution of the light chains in alkaline urea starch gel and polyacryl-
amide disc electrophoresis. Subsequent to these initial observations,
several antibodies have been examined in m uch g reater d eta il for
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SEARCH FOR
ANTIBODIES
WITH MOLECULAR UNIFORMITY
19
evidence of molecular uniformity (Eichmann
et al.,
1970a; Hood
e t
al.,
1969; Miller et al., 1967; Davie et al., 1968; Fleischman et at., 1968;
Braun a nd Krausc, 19 68 ). Observations
on
one
of these antibodies
will be reported in detail here.
Depicted in Fig. 9 arc microzone electrophoretic patterns of a
preimmune serum and a Group
C
antiserum R27-11 before and after
absorption with Group C carbohydra te (Eichmann
e t
al., 1970a) . The
sharp monodisperse component contained 36 mg./nil. of y-globulin; 93%
of this is precipitable with the C carbohydrate. This monodisperse anti-
body component was isolated from the antiserum by preparative agarose
electrophoresis. Such a preparative run is also depicted in Fig. 9. Only
the values for the protcin eluted from the block in the region of the
FIG.
9.
On the left, microzone electrophoretic patterns of preiinniune serum
and antiserum from rabbit R27-11 after 4 wecks immunization with Croup C
vaccine. Th e pattern of this serum after absorption
(abs) with Group C carbo-
hydrate (CHO) is
also
shown. Below the microzone patterns are the densitonietric
patterns of the immune serum before and after absorption with carbohydrate. Th e
shaded area in the lower diagram represents the antibody absorbed from the anti-
serum. On the right, preparative agarose electrophoresis of antiserum R27-11 (solid
line) and, for comparison, the same electrophoresis of a human serum from a patient
with multiple myeloma. (Adapted from Eichmann
et
al., 1970a.)
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20 RICHARD M. KFiAUSE
7-globulin are shown. For comparison, there is shown also the pattern
of serum of a patient with multiple myeloma. Fractions of the block in
the region
of
the peak component were pooled and concentrated. This
material was employed for subsequent studies which indicate molecular
uniformity.
In Fig. 10 are the 9.4M urea disc electrophoretic patterns of the fol-
lowing partially reduced and alkylated proteins. [These gels were
specially prepared to resolve the light chains (Reisfield and Small,
1966).] Gel 1 contains normal rabbit 7-globulin; gel 2 contains a Group C
antibody for which there was evidence for only partial restriction in
heterogeneity; gel
3
contains the very uniform antibody R27-11 isolated
by preparative electrophoresis
as
described in Fig.
9;
gel
4
contains
human Bence-Jones protein. The densitometric tracings of these gel
patterns are depicted in Fig. 11. Normal rabbit light chains are usually
distributed in eight to ten bands. The antibody with only partial restric-
tion of heterogeneity yields light chains that resolve into four distinct
bands, The very restricted antibody R27-11, depicted in Fig. 10, has one
major band which contains at least
90
of the total light-chain protein.
FIG.
10.
Light chain patterns. Polyacrylamide gel disc electrophoresis, pH 6.74,
in 9.4 M urea
of
reduced and alkylated y-globulin preparations: geI 1, normal rabbit
y-globulin; gel 2, a Group C antibody with only partially reduced heterogeneity;
gel
3,
peak component
of
antiserum
R27-11;
gel 4,
a
human Bence-Jones protein.
The direction of migration is from th e bottom to the top. (From Eichmann
et
aE.,
1970a.
)
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21
E A R C H FOR ANTIBODIES W I T H M O L E CU L A R U X I F O R M I T Y
heavy c h t l i g h t chains-q
t-heavy ch -- tc -l ig ht chains-1
ZO
0
1
klorge-+ small pore gel_____
FIG.11. Densitometric tracings of the disc electrophoretic patterns depicted in
Fig. 5. Gel
1,
normal rabbit y-globulin; gel
2,
a Croup
C
antibody with only partially
reduced heterogeneity; gel 3, peak component of antiserum R27-11; gel 4, a human
Bence-Jones protein. The direction of migration is from the left to the right. (From
Eichmann et al., 1970a.)
This pattern is indistinguishable from that obtained with
40
p g .
of
a
human Bence-Jones protein.
The disc electrophoretic gels, constructed to resolve the heavy
chains, for two partially reduced and alkylated antibodies are depicted
in
Fig.
12.
In these gels, the light chains have migrated beyond the gel
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22
RICHARD M . KRAUSE
FIG.
12. Heavy chain patterns. Polyacrylamide disc electrophoresis, pH 6.74 , in
9 . 4 M urea of reduced and alkylated y-globulin preparations: gel 1, a Group
C
antibody with only partially reduced heterogeneity; gel 2, peak component
of
antiserum R27-11. The direction of migration is from
the
bottom to the top. ( F r o m
Eichmann et
al.,
1970a.)
and are not seen in the photograph. Heavy chains of a heterogeneous
antibody were resolved into ten bands, whereas the heavy chains of
the very restricted antibody R27-11, described above, were confined to
only four bands. Two of these bands appear prominent. Similar patterns
may be seen with the heavy chains of a myeloma protein (Dorner et al.,
1969).
The case for uniformity of streptococcal antibodies which are mono-
disperse by electrophoresis is further supported by the demonstration
that they have individual antigenic specificity. Experiments similar to
that depicted in Fig. 2, which demonstrated individual antigenic specific-
ity
of
human antibodies to blood Group
A
substance were also per-
formed with the streptococcal antibodies (Braun and Krause,
1968).
In
all of these studies, the anti-antibodies have been prepared in goats.
The view that the antigenic specificity of these streptococcal anti-
bodies is associated with the Fab fragment was substantiated by immuno-
electrophoretic analysis which employed papain digests of a mono-
disperse antibody isolated by preparative agarose electrophoresis. Such
an analysis is depicted in Fig. 13. The papain digest of rabbit pooled
Fraction I1 gave two arcs with unabsorbed specific anti-antiserum. The
precipitin arc toward the cathode is formed by the Fc fragment, and the
arc toward the anode is formed by the Fab fragment. The situation
is
reversed for the precipitin arcs of the Fab and Fc fragments of the
streptococcal antibody employed here. This is because this antibody
exhibited a slow migration in electrophoresis and traveled a greater
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23
EARCH FOR ANTIBODIES
WITH
MOLECULAR UNIFORMITY
FIG. 13. Imniunoelectrophoresis analysis of Fa13 and Fc fragments of a Group
A-variant antibody with very restricted electrophoretic heterogeneity isolated from
antiserum by preparative electrophoresis. Well 1, papain-digested pooled rabbit
Fraction 11; well
2,
papain-digested isolated Group A-variant antibody. Trough a,
unabsorbed goat anti-antibody to the Group A-variant antibody; trough b, the same
anti-antibody absorbed with Fc fragments from pooled rabbit Fraction
11.
Trough c,
the same antibody absorbed with Fraction 11. (From Braun and Krause,
1968.)
distance toward the cathode than the bulk
of
the normal y-globulin in
pooled Fraction 11. This interpretation was clarified by the results of
immunoelectrophoresis in which either anti-antibody absorbed with Fc
of pooled Fraction I1 or anti-antibody absorbed with pooled Fraction
11
was added to the troughs. When the anti-antiserum absorbed with Fc
was added to the trough, only the Fab fragments of both the antibody
and Fraction I1 gave precipitin arcs. When anti-antiserum absorbed
with pooled Fraction I1 was added to the trough, the Fab fragments of
only the antibody gave a precipitin arc. No reaction occurred between the
Fab fragment of pooled Fraction
I1
and this absorbed anti-antiserum.
Taken together, such experiments indicate that individual antigenic
specificity is a feature of the Fab fragment of this streptococcal antibody.
Absence of precipitin reactivity between the light chain of this strep-
tococcal antibody and the anti-antibody absorbed with Fraction I1 in-
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24
RICHARD M.
KRAUSE
dicates that the light chain alone does not determine the individual
antigenic specificity. Similar results have been achieved with several
other antibody preparations isolated by preparativc electrophoresis.
Among the indirect criteria that have been used to judge the struc-
tural uniformity of antibodies, individual antigenic specificity is perhaps
the one which is most indicative of
a
homogeneity similar to that ob-
served for the myeloma proteins. Such a consideration stems from the
fact tha t the antigenic site of antigenic individuality includes the hyper-
variable region of the y-globulin molecule.
The
case for individual antigenic specificity as
a
criterion for the
molecular uniformity of an antibody is considerably strengthened when
it can be shown that all, or at least
a
very major portion, of the antibody
molecules react with the specific anti-antibody. Such a result has been
show n in the following experiment. Aliquots of several antibody pr ep ara -
tions, isolated by preparative electrophoresis, were labeled with
l';I
and
precipitated w ith a n excess amo unt of th e homologous anti-antiserum
which had been absorbed at equivalence with Fraction
11.
Controls em-
ployed two absorbed heterologous anti-antisera. The proportion of the
radioactive antibody in the precipitates and the supernatants were cal-
culated as per cent of the total radioactive antibody used in the test. The
results for antibody fractions from antisera R23-61, R22-79, and R27-11
are shown in Fig. 14 (Eichmann e t al., 1970 b). Th e antibody from
antiserum R27-11, already discussed in detail above, showed an excep-
tional degree of uniformity as judged by several criteria including dis-
tribution of genetic markers (Kindt et al., 1970c) and N-terminal amino
acid sequence of the light chain (Eichmann et
al.,
1 9 7 0 4 . Of this anti-
body preparation, 89% is precipitated by its individual anti-antiserum. A
similar result is achieved with the fast fraction of antiserum R22-79.
Since these antibody preparations were obtained by preparative electro-
phoresis alone, t he y contain approximately
10
to 15% onspecific 7-globu-
lin, which does not react with the absorbed anti-antibody. Only 70%
of the slow fraction of antiserum R22-79 was precipitated by the anti-
antiserum to the slow fraction. This result is consistent with other experi-
mental data pointing to a certain degree of heterogeneity of this antibody
component. I t is app aren t that experiments such
as
this afford an addi-
tional means to estimate th e uniformity of an antibody preparation.
An important characteristic of myeloma proteins is the selectivity
they show with respect to gene expression. Studies with the human allo-
typic markers (G m an d Inv types) have shown further tha t only one of
two possible alleles for both the L and the H chains are expressed in
the myeloma proteins in individual heterozygotes with respect to these
markers (M artensson, 1 96 1). Finally, only one of t he m any possible
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SEARCH
FOR ANTIBODIES
WITH MOLECULAR
UNIFORMITY
25
1 9 9
Goat ant i-
ant ibody absorbed
wi th pooled
rabb i t F rac t ion I I
89 8
R 22-79
Ant i - fas t
Fx
R 22-79
Ant i - s low Fx
R
2 3 - 6 1
Anti-slow Fx
R 2 7 - 1 1
Ant i - fas t
Fx
The Proport ion of an Ant ibody Preparat ion Prec ipi tated by
Homologous aiid Heterologous Ant i -ant isera
Per cei i t ant ibody recovered i n prec ip i tate and supernatant
Ant iserum R 22-79
Slow
Fx
~
Ppt.
Supt . Pp t .
Supt .
% " l o
% a
91
8
2 9 7
4
9 8
70
3 1
3 9 5
2 1 0 0
Ant iserum R 2 3 - 6 1
Slow
Fx
2 2
Ppt . sup t .
'/.
70
4 9 5
80
2 1
Ant iserum R 2 7 - 1 1
Ppt . Supt
70 %
2 9 9
1
9 9
FIG.
14.
Per cent of rabbit antibody recovered in the precipitate (ppt. and
the
supernatant (supt calculated from the radioactivity. Each antibody preparation
was labeled with
'Y.
All
precipitin tests done with an excess
of
anti-antibody. (From
Eichmann et al., 1970b.)
variants of each of these chain types is expressed (Hilschmann and
Craig, 1965). A similar selectivity of expression was observed in some
of
the steptococcal antibodies formed in response to immunization with
the streptococcal vaccine.
The allotype of the homogeneous antibody R27-11, which has been
described in considerable detail thus far, is shown in Table I1 (Kindt
et al., 1970~).
his rabbit was homozygous with respect to both the
group a and group b allotypes. In the purified antibody, the
H
chain is
selected from the population not possessing the allotypic marker from
group a. In effect, no purified antibody was precipitated by antisera to
the a1 marker. Oudin (1961) has shown that rabbits normally contain
some 7-globulin moleculcs lacking the group a allotypic specificities.
David and Todd (1969) have shown that it is possible to obtain rabbits
devoid of imniunoglobulin with the group a allotype by embryo transfer
into does that produce antibody to the allotypic specificities genetically
present in the transferred embryo. The absence of group a allotypic
specificities in this uniform streptococcal antibody suggests that it is
possible to obtain specific antibodies selected from this population which
lack the allotypic marker just as individual myeloma proteins are believed
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26
RICHARD
M. KRAUSE
T A B L E
I1
IgG
U Y
ALLOTYPIC
ANTISERA"
PRECIPITATIONF R A D I O - I O D I N A T E D STREPTOCOCCAL GROUP
c
Awrrim)Y
A N D
Precipitation by ant,isera
( )*
Rabbit Allotype Sample a1 b4 F C
R27-11
al , b4 Preimmune IgG 6 2 93 97
Electrophoresis preparationc 10 99 92
Group
C
antibody
Purified antibodyd 1 99 98
Notiantibody I g G 62 97 96
From Kindt et al. (1970~).
b Determined by coiintiiig 12hI i n the precipitat,e.
c
Isolated
as
depicted in Fig.
9.
d
Recovered from the electrophoretic antibody preparation by means of a Sephadex
e
The nonantibody
IgG
in the electrophoretic antibody preparation which is
not
G-200 column which serves as a specific immunoabsorbent for this antibody.
reactive with Sephadex in the group-specificC carbohydrate.
to result from enhanced production of an otherwise normal y-globulin
molecule. In additional studies with other purified streptococcal anti-
bodies, which were isolated from a double heterozygous rabbit, a
selective absence of
a
marker at both the a and b loci was observed.
For example, from a rabbit which was typed a l , a3, b4, b5, A l l , A12, the
isolated Group C antibody was a l , b4, A12 (K ind t et al., 19 70 a). This is
consistent with previous work which has shown that group a allotypes
share chains with A l l an d A12 determinants (Pra hl
et
al . , 1970) , and
these two groups of allotypes are linked genetically (Kindt et
al.,
1970b;
Zullo
et
al., 1968). Recently, Rodkey
et
nl. (1970) have isolated anti-
bodies to streptococcal carbohydrate by electrofocusing. These anti-
bodies, although isolated from a heterozygous rabbit, had
a
single
allotypic specificity at both the a and b loci.
The case for molecular uniformity
of
antibody R27-11 is considerably
strengthened by the amino acid N-terminal analysis of the light chains.
In Table I11 are presented the results of the quantitative three-cycle
Ed m an analysis of th e amino acid alternatives a t the first three N-terminal
positions. The amino acid d ata from antibody R27-11 were com pared to
those of the preimmune y-globulin of the same rabbit. Calculation of per
cent yield and per cent recovery are also given in Table 111. The da ta
for preimmune 7-globulin an d antibody R27-11 were derived from t he
amino acid analyzer chromatograms
of
the hydrolyzed P T H amino acids.
In the case of an tibody R27-11, th e yields of a single predominant amino
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
27
T A BL E
111
AM I NO
.4c11)s
N-TERMINALEQUENCEOSITIONS, 2, A N D
3
FOR
THE
LICIITCHAINS
O F
PREIMMIJNE
R A I ~ I T
- G I . O I I U L I N
N D
FOR
ANTIBODY
TO GR0t.P
c 8TREPTOCOC'CAL CARR0HYI)RATE
FROM R A R H I T7- 11~~ '~
Position
I
Position 2 Pasition
3
Itecov-
Tield
ery
Light chains
( ) 06)
Recov-
Yield
ery
7;) ( )
Asp
14
Preitnmiine -,-globidin
26
Asp 85
Val 4
Leii 4
Gly
8
lo
a Quantitative Edmm procediire employed. See footnote a to Table I for discushion
of the
calculation.
From
Eichrnann
d
al.,
1970a.
acid a t each of th e first three N-terminal positions resembles those
for
the Bence-Jones protein (Hackney) which were given in Table I. I t is
obvious that there are multiple amino acid alternatives at the first three
N-terminal positions in the preimmune 7-globulin light chains. Recently,
th e light chains of Grou p C antibody K27-11 have been examined in
the sequenator.
A
single unambiguous amino acid residue was obtained
a t each of th e first fifteen N-terminal positions, an d the sequence is
listed in Table
IV
( H o o d
et
al., 1970 ). Comm ent on the sequence data
on the light chain of antibody from rabbit R24-61 will be reserved for
the Discussion.
The case for molecular uniformity for the antibody in antiserum
R27-11 can be summarized as follows: monodisperse distribution of
the antibody by microzone and preparative zone electrophoresis; mono-
disperse distribution of the light chains by disc electrophoresis; allotype
exclusion; and a single amino acid sequence for the first fifteen N-ter-
minal residues of t he light chains. In all respects by these various methods
of examination, this antibody is as homogeneous
as
a
myeloma protein.
It remains to
be
determined if a single amino acid sequence is observed
in the hypervariable rcgion of the light chains. Such studies are now
under way.
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TABLE 1V
COMPARISONETWEEN N-TERMINALMINOACID SEQUENCE
F
ALLOTYPE4 LIGHT CH
TO
STREPTOCOCCALARBOHYDRATES
ND
THE
AMINOACID RESIDUEAL
IN
THE K
LIGHTCHAINS
N
MAN
AND
MOUSE"
Rabbit and N-Terminal position
preparation type
0
1 2 3
4 5
6 7
8
9
antibody All*
R27-11
R24-6 1
Fast
;
peak component
b4(k)
Ala Asp
component b4(k) ( )
Ala
Man
Mouse
Glu Asp
Glu
Lys
ASP
Glu
Val
Phe
Ile
Ile
Ile
Val
M e t
Ile
Val
Thr
Val Met
Glx Met,
Val
Met
Val Val
Gln Met
Val
Leu
Leu
Val Met
Gln Val
Thr
Ile
Leu Leu
Thr Glu Thr Pro Ala
Thr
Glu Thr Pro
Ala
h4et
Thr Glu
Thr Gln Ser Pro Ala
Thr Ser
Leu
GlY
Thr
A S X
Thr Gln Ser
Pro
Ala
Thr Ser
Thr
Leu
Adopted
from
Hood
et
al. 1970.
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SEARCH FOR ANTIBODIES WITH MOLECULAR
UNIFORMITY
29
B. RABBITANTIBODIES
O PNEUMOCOCCAL
CAPSULAROLYSACCHAKU)ES
Intravenous immunization of rabbits with formalized pneumococci
has been employed by Haber (1970) and Pincus et
al.
(1970a,b) for the
gcneration of large quantities of antibodies with restricted heterogeneity.
Rabbits immunized with th e whole bacteria prod uce predom inantly anti-
bodies to the capsular polysaccharide. Pneumococci Types I11 and VIII
were used for these studies. Immunochemical considerations dictated this
choice. These are long-chain polymers an d th e sequence of th e rep eating
sugars is known. These sequences are d epic ted in Fig.
15.
Antibodies are
S
m,
.f-3)
-4
- D -
glcA-(l--4)-~
?-D-glc-(ff
S V I I ,
4)
-4 - D - g l c A - ( ~ - ~ ) - 4 - D - g l c - ( l ~ 4 ) - o - 0 - g l c - ( l ~ ) - ~ - D - ~ ~ l - ~ l ~
FIG. 15. Sequence of sugars in repeating subunits of pneumococcal poly-
saccharides. Types 111 (SIII) and VII (SVIII ) ; glc
=
glucose; glcA = gluciironic
acid; gal = galactose. (From Haber, 1970, adapted from Heidelberger, 1967.
)
directed against a limited portion of such a sequence. Octasaccharides of
these polysaccharides, for example, readily inhibit th e precipitin reaction,
an d when such fragments are tritiuni-labeled, they can be used for bind-
ing studies to determ ine association constants ( Pappenheiiner et
d.
968) .
A primary, secondary, and tertiary immunization schedule, similar
to that used for streptococci, was employed for pneumococcal immuniza-
tion of rabbits. Approximately 6 to
8%
of th e rabbits ha d a predominant
major antibody component in the antiserum. One of the Type VIII ant i-
bodies, isolated by immunoabsorbent methods to be described in the
next section, has several of these pro per ties of a myeloma protein which
are indicative of uniformity, T he light chains ar e distributed in one m ajor
band by disc electrophoresis; and a single amino acid sequence is ob-
served for th e first eleven N-terminal residues of th e light chains (W ate r-
field et al., 1970) .
The binding of the tritium-labeled octasaccharides derived from the
Typ e VI II carboh ydrate to the ant ibody to Type VIII was examined by
equil ibrium dialysis , and the data are depicted in the Fig. 16 (Haber,
19 70 ). T he association constant is 2.5
x
lo5,
a
range commonly seen
fo r antibodies to carboh ydrates a nd for th e antibodies to pneumococcal
capsular polysaccharides
(
Pappc'nheinier
et
al.,
1968) .
The
Sips plot
(Nisonoff an d Pressman, 1 95 8) yields a value for the heterogeneity index
which is within experimental error of unity. These homogeneous binding
data arc not necessarily indicative of homogcneous antibody as judged
by other criteria. Othcr antibodies
to
p n c ~ ~ m o c o c c a lolysaccharides
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30
RICHARD
M.
KRAUSE
0.2
0
-
0.2
- 0.4
LF 0.6
P
2 -0.8
4
-
1.0
-1.2
a
=0.98
I I I I I I I I I
-6.6 -64 -6.2 -6.0 -5.8 -5.6 -5.4 -5.2 -5.0
L o g
c
- 6.6 -6.4 -6.2 -6.0 -5.8 -5.6 -5.4 -5.2 - 5.0
L o g
c
FIG. 16.
Logarithmic plot
of
equilibrium dialysis data. Moles ligand bound per
mole of antibody assuming
a
molecular weight of 150,000; N, number of binding sites
per mole, assumed to be 2; C, concentration
of per
ligand; Ka, association constant;
and a, heterogeneity index as derived from a Sips analysis employing an SDS 940
computer (program
by
Marcia Stone). (From Haber,
1970.)
which are heterogeneous by electrophoretic criteria, for example, give
evidence of homogeneous binding (Pincus
e t
aZ., 1968). One possible
explanation for such data is that heterogeneous antibodies consist
of a limited selected number of sets of antibodies, all
of
which, however,
have a similar binding site.
C.
ISOLATION
ROM
ANTISERA
F
ANTIBODIES
O
BACTERIAL
ARBOHYDRATES
The recovery of antibodies with molecular uniformity from rabbit
antisera has been achieved by use of immunoabsorbents and prepara-
tive electrophoresis or a combination of both. The method to be em-
ployed is dictated by the electrophoretic character of the antibodies in
an antiserum. If all of the antibody is confined to a single major M-
type component, similar in appearance to a myeloma protein on elec-
trophoresis, then preparative electrophoresis can be used to isolate the
antibody, just as this method can be used to isolate myeloma proteins.
Most commonly, however, the problem is more complex than this. It is
for this reason that techniques
of
antibody isolation must be considered
in some detail here.
In Fig. 17 are depicted representative examples
of
the kinds of
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY 31
I
R
FIG.
17.
Representative examples
of
rabbit antisera to bacterial carbohydrates,
Precipitating antibody is indicated by the shaded area. a, An example of antiserum
with all
of the
antibody in a predominant
M
component; b and c, examples
of
anti-
sera
with
antibody in a monodisperse component
and
in a shoulder, broad com-
ponent; d, an example of an antiserum with antibody distributed in two distinct
components.
electrophoretic patterns of antisera to bacterial carbohydrates from
which antibodies are isolated. Such patterns have been seen for the
antisera of rabbits immunized with either streptococci or pneumococci.
In each example, the precipitable antibody is indicated by the shaded
area in the region of the y-globulin. To be noted, in each instance, is a
small portion (usually about
10
to 15%)of 7-globlin represented by the
unshaded area, which is not precipitable by the soluble antigen. This
nonprecipitable 7-globulin is the nonspecific y-globulin in the serum.
Therefore, recovery of the y-globulin from such an antiserum by means
of preparative electrophoresis aIone will not yield an antibody popula-
tion clearly devoid of nonantibody IgG. Nevertheless, recovery, by
preparative electrophoresis, of a peak component when it
is
a major
predominant one from a n antiserum such as example
a,
yields a remark-
ably honiogcneous antibody preparation which is adequate for most
purposes. Many of the studies reported in the previous section were
done with antibodies isolated in this
way.
Inimunoabsorbents are another powerful tool for the isolation of the
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32 RICHARD
M.
KRAUSE
antibodies from these antisera. Clearly, in the case of antiserum a
(F ig . 1 7 ) , either use of immunoabsorbents or recovery of antibody from
a
specific immune precipitate wo uld yield a n antibody prep aration th at
was essentially homogeneous because in such an antiserum all of the
antibody is located in one monodisperse component. But, immune re-
sponses, such as the one represented by antiserum A are uncommon.
More common are antisera b, c, a n d d which have more than one anti-
body component. Clearly, in these instances, reliance on immunoabsorb-
ents alone for the recovery of an antibody would yield heterogeneous
antibody preparations. In the case of antisera a and b, there is now a
good dea l of da ta which indicate th at th e predom inant monodisperse
antibody component contains antibody with molecular uniformity,
whereas, the antibody in the broad shoulder component is heterogeneous.
In such a case, recovery of
all
of the antibody from the antiserum would
yield a heterogeneous antibody population. Finally, in the case of anti-
serum c, in which there are two distinct antibody components (and,
on occasion, more than two), there is considerable evidence that each
peak has its own individual antigenic specificity (Braun and Krause,
1968) and a distinct amino acid sequence (Hood et al . , 1970). There-
fore, recovery of t h e antibody from such a n antiserum b y means of an
immunoabsorbent would result in an antibody preparation which is
clearly heterogeneous.
The conclusion to be drawn from these remarks is that the method
selected to isolate antibody from a particular serum depends upon a
careful evaluation of the number of major antibody components in the
antiserum as judged by microzone electrophoresis. It is obvious that a
combination of both preparative electrophoresis and immunoabsorbents
has the potential of yielding antibody that has a very marked restriction
in heterogeneity and devoid of nonantibody y-globulin.
Several different immunoabsorbents have been successfully employed
to recover the antipolysaccharide antibody from antisera. The specific
immunoabsorbents fo r the antibodies to T ype
111
an d V III pneumococcal
polysaccharides, as described by Haber
(1970) ,
were synthesized by
a
two-step procedure. The polysaccharide was coupled to bovine serum
albumin by the method of Avery and Goebel (1931) and the amino
groups of the protein were then reacted with bromacetylcellulose (Rob-
bins
et
al., 1967). By use of the specific immunoabsorbent, 95%of either
the Type 111 or VII I polysaccharide antib ody was recovered from th e
antisera. Recovery was achieved by a batch process in which all of
the absorbed antibody was eluted. This method for isolation of the
antibody has the advantage over preparative electrophoresis in that all
of the recovered protein is antibody to the carbohydrate. There is no
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SEARCH FOR
ANTBODIES
WITH MOLECULAR UNIFORMITY
33
residual nonspecific y-globulin in the isolated antibody as is the case
when preparative electrophoresis is employed. However, this immuno-
absorbent method of preparing uniform antibody is applicable only to
antisera such as example a depicted in Fig. 17. Obviously, batch rc-
covery by immunoabsorbents of all the antibody in antisera, such as
examples b, c, and d (F ig . 1 7 ), will yield antibodies with varying degrees
of heterog eneity, and , in th e case of example d, th e antibody preparation
will have at least two distinct antibody populations.
Two recent innovations suggest technical procedures for circumvent-
ing the difficulties described thus far in isolating from an antiserum a
single antibody population devoid of nonspecific y-globulin. Recently,
Parker and Briles (1970) and Eichmann and Greenblatt (1970) have
described the fractionation of antibodies from streptococcal antisera by
means of affinity chromatography.
The antipolysaccharide antibodies were purified from
a
streptococcal
Group A antiserum and simultaneously fractionated into electrophoreti-
cally distinct components on the basis of their affinity for the phenyl-
,8-N-acetylglucosaminide group. p-N-Acetylglucosamine is the terminal
immunodominant determinant of Group A polysaccharides. The immuno-
absorbent was prepared by coupling p-aminophenyl-P-N-acetylglucos-
aminide to Sepharose activated with cyanogen bromide (Axen
et
al.,
1967; Porath e t al., 19 67 ). Essentially all of th e an tipolysaccharide anti-
bodies in the antiserum were absorbed on a column of this immuno-
absorbent, while other serum components passed through. Specifically
absorbed antibodies were eluted with a gradient of N-acetylglucosamine
from 0.0 to 0.3 M a t neutral pH . Two distinct antibody components were
recovered each at a different amino sugar concentration, and each
possessed a distinct electrophoretic mobility. Light chains from these
two
antibody components had one major band on disc electrophoresis.
An immunoabsorbent column for the antibodies to Group C carbo-
hydrate were prepared by coupling the whole carbohydrate to the
Sepha rose instead of th e p-aminophenyl-a-N-acetylgalactosaminide
( Eichmann an d Greenblatt , 19 70 ). Th e whole carbohydrate w as em-
ployed because of the difficulty in preparing the a-N-acetylgalactos-
aminide compound for use in
a
specific Group C immunoabsorbent,
similar to the one for Group A. In order to couple the Group
C
carbo-
hydrate to activated Sepharose, the antigen was partially deacetylated
(Axen et
al.,
1967; Porath et al., 1967; Kristiansen et al., 1969) . A
column, employing this immunoabsorbent, completely removed the pre-
cipitating antibody to the Group
C
carbohydrate from the antiserum.
By elution with a p H and sal t gradient s tart ing with 0.1 hl phosphate
bufTered saline at p H 7.2 an d end ing with
1M
sodium chloride-0.5
M
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34 RICHARD M. KRAUSE
acetic acid at pH
2.5,
several distinct antibody components were eluted.
Each component possessed a distinct electrophoretic mobility. Such
an experiment is depicted in Figs.
18
and
19.
A microzone electrophoretic pattern
of
a Group C antiserum, prior
to absorption by an immunoabsorbent is shown in Fig. 18. The antiserum
after removal of the antibody by passage through an immunoabsorbent
column described above is also shown. Clearly, the bulk of
t h e
y-globulin
is specific antibody and has been absorbed onto the column. The elution
Group C ant iserum
Unobsorbed t o column
: \
Fr I off column
ll.?L--
F r
IU
off column
FIG.
18.
Microzone electrophoretic patterns
of
a
Group
C
antiserum before and
after absorption b y an immunoabsorbent column described in the text. The lower
three patterns are antibody fractions eluted from the column by means
of
a pH
gradient (shown in Fig.
18).
Fraction I (Fr I ) , Fraction I1 (Fr I I ) , and Fraction 111
(Fr
111)
refer
to
the corresponding fractions in Fig. 18.
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
35
Elu t ion
o f
ant ibody absorbed
lo
immunoabsorbent column
by pH grad ien t
7
1 2
6
- 10
E
2 0 8
v
06
5
\
4
c
C
V
0 4
2
11
0 2
0
4 12 2 0
2 8
36 4 4 5 2
60
68
7 4
Tube number
FIG. 19.
Elution of antibody fractions from the Group C antibody absorbed
onto a specific immunoabsorbent column by means of a
pH
gradient. Fraction
I,
which was eluted with
a
slight lowering of pH, has the most rapid electrophoretic
mobility (see Fig. 18),whereas Fraction
11,
which was eluted with a greater lowering
of
pH has
a
slower mobility (see Fig.
18).
of the antibody from the column by a pH gradient is shown in Fig. 19.
Three protein fractions, Fraction
I, 11,
and 111, were resolved. The tubes
containing each fraction were pooled, and the pools, after concentration,
were examined by microzone electrophoresis. The microzone patterns of
these fractions are also shown in Fig. 18. This microzone analysis was
performed simultaneously with the unabsorbed and absorbed antiserum.
It
is, therefore, possible to identify an isolated fraction as a particular
one
of
the multiplc antibody components in the unabsorbed antiserum.
Antibodies have b een recovered from m ore tha n six antisera, and , in each
case, the antibody with the most rapid electrophoretic mobility is eluted
with the least fall in the pH of the buffer, whereas, the antibody com-
ponent with the slowest mobility is eluted with the greatest fall in
buffer pH.
Fraction
I1
recovered by the gradient elution from the immuno-
absorbent column is very restricted in electrophoretic mobility and the
light chains migrate in one prominent band by disc electrophoresis.
Although much remains to be done to determine the efficacy of these
immunoabsorbents and the resolving power of the gradient elution
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36
RICHARD M. KRAUSE
technique, it would appear now that at least one, and in some cases two
or three, restricted antibody components can be recovered from a single
antiserum by these methods.
As
a consequence, antisera such as examples
b,
c, and d (Fig. 17) may be just as useful as sources for antibodies with
uniform properties as is the much more rare “monoclonal” antiserum,
example a.
A
special case of affinity chromatography has been particularly useful
for the isolation of uniform antibodies from occasional Group C strepto-
coccal antisera. This is based on the fortuitous observation that the anti-
bodies to Group C carbohydrate in these antisera bind weakly to Sepha-
dex (Kindt et al., 1970~) . ecause of this affinity, the specific antibody
is sufficiently retarded on the column
so
that it can be separated from
the nonantibody components that pass through more readily.
A
high
degree of purification of an antibody was achieved with a Sephadex
column, and
the
results
of
the procedure are depicted in Fig. 20. A Group
C antibody preparation was recovered by preparative electrophoresis
from antiserum R27-11 (Fig. 9). Such a preparation contained approxi-
mately 85 to 9% specific antibody and 10 to 15%nonspecific y-globulin.
This preparation was passed through the Sephadex
G-200
column. The
nonspecific 7-globulin was eluted in the same effluent in which radio-
labeled y-globulin was recovered and had no antibody activity for the
*
1.5
-I
* I i ‘,
FRACTION
FIG.20.
Isolation of Group C antibody from rabbit R27-11 by chromatography
with a Sephadex
G-200
column.
A
5.0-mI. sample containing
0.10
mg.
of radio-
iodinated “‘I antibody isolated from antiserum by electrophoresis was mixed with
25 mg. of normal IgG and was applied
to
a column (90 X
2.5
cm.) of Sephadex
G-200 in
0.1 M ,
H 6.8, potassium phosphate buffer.
( X -
-X O.D., 280; 0--0)
radioactivity. Fraction size was 8.0 ml.
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37
EARCH FOR ANTIBODIES WITH AfOLECULAR UNIFORMITY
Group C carbohydrate. All antibody activity was in the retarded ma-
terial. The quantitative allotypic data recorded in Table I1 indicate that
the antibody e luted in the retarded volume is devoid of th e a 1 mark er,
whereas, about 10 of the antibody preparation obtained by electro-
phoresis alone, prior to passage through the Sephadex column, was
precipitated by anti-a1 antiserum.
D. FACTORSNFLUENCING
HE
OCCURRENCE
F
HIGHANTIBODY
The occurrence
of
one prominent monodisperse antibody component
in the antisera of rabbits that have been immunized with bacterial
vaccines has in large measure remained unexplained. Undoubtedly,
a
number of variable factors, as yet undetermined, influence this occur-
rence. Factors which undoubtedly play
a
role include: the route of
immunization, the physical state of the antigen, prior sensitization, and
the genetic backgro und of the rabbit.
There is little or no published information on the most effective
composition of the vaccine for the production of potent antisera for
streptococcal grouping. Clearly, the size of the particles in the vaccine
may be important. Whole streptococci with the carbohydrate as t he
outermost cell wall element appear to stimulate
a
greater immune
response than isolated cell walls which on the dry weight basis are one-
fifth the size of intact streptococci ( McCarty, 1970). Furthermore, the
purified soluble carbohydrate which has a molecular weight between
8,000 and
10,000
is not antigenic in rabbits (Lancefield, 1970).
The possible advantages and disadvantages in alternative routes or
methods of immunization with bacterial vaccines has not been examined
in detail, at least not in recent years. The early impressions acquired by
Lancefield (1 97 0) were tha t alternative routes an d the use of adjuv ants
were not
as
effective and, perhaps, were much less effective, than intra-
venous immunization with vaccines composed of heat-killed streptococci.
It
is
possible that the potential advantage which can be achieved with
frequent intravenous injections has bcen overlooked because of the
magnitude
of
th e effort to administer antigen in this w ay. But, to seasoned
clinicians wh o recall patients with s ub acu te bacterial endoc arditis, prior
to thc days of antibiotics, who had prolonged persistent bacteremia, the
profound hypergammaglobulinemia in
the
rabbits immunized intra-
venously comes as no surprise.
Histological examinations have been done on rabbits killed at the
time the serum contained high concentration of uniform antibodies
( Haber, 1970; Braun an d Krause, 1 96 9). Inte nse proliferation of plasma
cells is seen in spleen, lungs, and lymph nodes. By use of immunofluores-
RESPONSES
ITH
RESTRICTEDETEROGENEITY
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38 RICHARD M . KRAUSE
cence, Hab er (19 70 ) observed tha t the majority of the plasma cells in the
spleen of rabbits immunized with Type VIII pneumococci were making
antibodies to the T yp e VIII carbohydrate. Although m uch remains to
be
learned about the cellular events that occur with intravenous immuniza-
tion, it would appear that this is a very effective way to stimulate plasma
cell proliferation and, as
a
result, achieve high antibody responses.
Additional information on antibody formation in rabbits immunized with
pneumococcal vaccines can be found in Hu mp hrey and Suli tzeanu (1957 )
and in Askonas and Humphrey (1957, 1958).
Rabbits which do not respond with a uniform population of antibodies
or with a high antibody level after primary immunization, may do
so
after second immunization. In some cases, the increase in hypergamma-
globulinemia af ter second immunization ca n b e striking. As a n examp le,
the antibody level in one rabbit after first immunization was only 3
mg./ml. After a second immunization, the antibody level was 50 mg./ml.,
an d in this case there was evide nce for molecular uniformity of th e
antibodies. Although the explanation for this phenomenon
is
obscure,
it is conceivable that intense immunization over a prolonged period
selects a restricted population of cells which undergoes proliferation. In
this connection, there is an intriguing parallel between these uniform
populations of antibodies in rabbits after second immunization and the
development of a myeloma-like condition in mink with Aleutian disease.
It was observed that late in the course of Aleutian disease, some mink
showed a transition from a heterogeneous hypergammaglobulinemia to a
homogeneous myeloma-like hypergammaglobulinemia. Such a finding
suggests the ascendancy of a few predominant clones of plasma cells
(D. D.
Porter e t
al.,
1965).
I t remains to b e clarified with certainty wh y some rabbits h ave
a
high
immune response following intravenous immunization with streptococcal
vaccines, whereas the majority respond poorly or with onIy modest levels
of antibody. The possibility that such a response is under some form of
genetic control was investigated by selective br ee di ng of high-response
an d low-response breeding pairs. T he imm une response of th e offspring
was measured after immunization similar to that administered to the
parents (Braun et al., 19 69). Reported in Tab le V a re th e precipitin
levels to Group C carbohydrate in primary and secondary response
antisera for both parents and their offspring. The data suggest that the
mag nitude of th e imm une response is genetically transm itted. On e breed-
ing pair had a relatively low antibody response and so did their offspring.
Th e other breedin g pair had a relatively high antibody response an d
their offspring had similar responses. Although there is some evidence to
suggest that the antibody in the high responders is more likely to have
restricted heterogeneity than the antibody in the low responders, this
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
39
TABLE
V
C O N C E N T R A T I O N
OF
P R E C l P I T I N s TO G R O U P
c
C A R B O H Y DR A T E I N THE
ANTISERA
OF
P A R E N 'I 'A I ,
H I G H
t E S P O N S E A N D
L O W
RESPONSE
a u s r T s A N D
THEIR
O F F S P R I N G I M MI JN IZ E I) W I T H G R O U P
c
VACCINE''
Colic. of
Low response breeding pairh High response breeding paiP
precipitins
(nig. ab/nil. Parents Offspring Parents Offspring
of
antiserrun) 1" 2" 1" 2" 1"
2" l o 2"
1
2
3 F
4
F
5 &I
6
7
8
9
JI
10
11
12
13
14
1
.i
16
17
18
22
23
2 3
29
30
31
32
3.5
37
38
42
45
48
55
6 2
2
3
1
1
2
1
1
-
-
6
5
1
1
2
4
3
1
3
2
1
1
1
1
1
1
1
1
1
1
1
1
1
2
1
2
2
1
1
1
1
1
1
19
a Not all offspring survived the interval between primary and secondary immutiiza-
tion. Therefore, the total number for second imniniiization
is
less t h a n the total for the
first immunization. Primary response antisera collected a t the end of 4 weeks of immuni-
zation. Secondary response antisera collected at the end of
3
weeks
of
immunization.
Interval between primary and secondary, 4 months.
*
F
=
female;
RI = male; 1" = primary itnniuriization;
2'
= yecoiidary immuni-
zation.
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40 RICHARD M.
KRAUSE
matter is not yet settled. Solutions to these genetic questions will be more
readily achieved if responses of this type can be reproduced in inbred
mice. Such studies are now under way.
No attempt will be made here to review the literature on the influence
of genetic factors on the immune response. This subject has recently been
covered by McDevitt and Benacerraf (1969) in the preceding volume of
this series.
E.
Selective recovery of a subpopulation of antibodies to the specific
C-terminal sequence of myoglobulin has been achieved by selective
elution from specific immunoabsorbents. Myoglobulin is a complex
protein, and rabbits immunized with a Freunds adjuvant preparation of
this protein produce antisera that contain multiple antibody populations
with specificities for different structural features of the molecule. Givas
et
ul.
(1968) isolated antibodies to the C-terminal heptapeptide of
myoglobulin by elution of the antibody from the immunoabsorbent
column with a synthetic heptapeptide identical to the C-terminal hepta-
peptide of myoglobulin. The antibody eluted with the synthetic hepta-
peptide was monodisperse by disc electrophoresis, and this is in contrast
to the polydisperse character of the total myoblobin antibody component
in the rabbit antisera. Yield of such restricted antibody, however, was low.
These studies on the restricted heterogeneity of antibodies to the
C-terminal heptapeptide of myoglobin are reminiscent of the restricted
heterogeneity of antibody to angiotensin which has been examined by
Haber e t ul. (1967) . Angiotensiii is an octapeptide. The immunizing
antigen was a branched chain polymer of angiotensin on poly-L-lysine.
The peptide was coupled via its carboxyl terminus to the E-amino groups
of the poly-L-lysine. The results of binding experiments with nonfrac-
tionated antiserum and iodinated angiotensin gave points that fell on a
straight line plot. The Sips analysis yielded an association constant of
2.64 x los L/ M
and a heterogeneity index within experimental error of
unity. Such data point to a uniform antibody with respect to binding
affinity, but there are no data available to indicate if such antibodies have
structural homogeneity.
The disadvantage in these methods of obtaining antibody with
molecular uniformity, by employing immunization with natural materials
other than the bacterial polysaccharides, is that yields of antibody are
small and the quantity is insufficient for extensive examination of amino
acid sequence. These studies with antibodies to angiotensin have but-
tressed the notion, however, that an important cause of antibody hetero-
geneity is antigenic heterogeneity and that reducing the heterogeneity
ANTIBODIES
O
MYOGLOBIN
ND ANGIOTENSIN
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
41
of an an tigen reduces t he func tional an d molecular hete roge neity of
the antibody.
F.
ANTIBODIESO SYNTHETIC NTIGENS
In the past several years, evidence has been accumulating from a
number
of
sources that antibodies with restricted heterogeneity may
be
generated in response to immunization with synthetic antigens. Both
optimism and enthusiasm were sparked by the observation of Nisonoff
et
ul.
(1967)
that a single rabbit produced a remarkably homogeneous
anti-p-azobenzoate ant ibody. The rabbit had been immunized with
p-aniinobenzoic acid coupled to bovine y-globulin, emulsified in com-
plete Freunds adjuvant. The specific precipitin antibody level reached
6 mg./ nil. of antiserum an d the antibody , purified from a specific immune
precipitate, crystallized out at a concentration of 60 mg./ml. in NaCl
bora te buffer , p H 8, ionic strength
0.16.
Th ere was
a
selective expression
of the allotypic markers in the antibody. The whole antiserum was allo-
type a l ,
a2,
b4; the dissolved antibody crystals were simply allotype al ,
b4.
It would appear that this remarkable antibody
is
the result of an
uncommon event because such antibody was not seen in numerous other
rabbits immunized with this antigen. How ever, it is possible, th at scrutiny
of
many of these antibenzoate antisera by microzone electrophoresis and
sub sequ ent fractionation of antibody components from the m by the
methods described above would havc yielded antibody preparations
which approached the uniformity of the one crystallizable antibody from
the one rab bit. This interpretation receives some s upp ort from th e subse-
quent studies of Daugharty et
ul.
(1969) on th e idiotypic characteristics
of the antibenzoate antibodies in many different antisera. The findings
that only a portion of an antibody preparation is immunogenic in allo-
typically matched rabbits a n d that t he sam e subfraction of th e antibody
is immunogenic in different rabbits sugest that the immunogenic popula-
tion is composed of a limited number of homogeneous groups of antibody
molecules.
The s tudies of Roholt e t
ul.
(1970) also suggest that antibodies of
limited heterogeneity may occur in antisera of rabbits immunized with
azo-p-beiizoate-bovine -y-globulin. Ra bbits were immunize d intravenously
over a period of several months to 2 years, and antisera were obtained
throughout this time. Antibody concelltrations ranged from 0.5 to 2
mg ./ml. The antibodies from
2
of 9 rabbits have several properties which
indicate restricted heterogeneity. The light chains were resolved into one
band by polyacrylamide disc electrophoresis. The antibodies were ho-
mogeneous with respect to their binding constants, with heterogeneity
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42 RICHARD M. KRAUSE
indices of 1.0, whereas an antibody with polydisperse light chains showed
gross heterogeneity of binding constants.
Taken together, these several reports on rabbit antibodies to azo-
benzoate groups suggest that they, too, may be a useful source of anti-
bodies with limited heterogeneity. The obstacle at the moment would
appear to be the relatively low concentration of these antibodies in the
serum. It is conceivable, however, that the immune response can be
enhanced through manipulation of the immunization procedures and
selective breeding of the rabbits.
One final approach which is employed to generate antibodies with
restricted heterogeneity will be discussed here. This is concerned with a
reduction in the degree of DNP antigen heterogeneity. In most studies
with hapten-protein conjugates, the molecules are more or less attached
randomly to the variable surface of the protein. With the thought that
such random attachment of the determinants may contribute significantly
to the heterogeneity of the antibody response, Brenneman and Singer
(1968) constructed a special antigen in which the DNP was attached to a
single characteristic site of a molecule. Papain was selected
so
that
advantage could be taken of the single SH group per molecule. The SH
group, available by activation
of
the enzyme, was reacted with a - ( N -
iodoacetyl)
L-
(
N-2,4-dinitrophenyl)-1ysine. The final product had one
DNP lysine group per molecule of papain. Rabbits and mice have been
immunized with this antigen. Although the yields of antibodies have
been very low, 5-1m of the animals produce antibodies which, by two
criteria, have restricted heterogeneity. These antibodies are resolved into
one major band by polyacrylamide electrofocusing. Normal y-globulin
resolves into thirty-five or forty bands (Trump and Singer, 1970). The
light chains are distributed in one major band by polyacrylamide disc
electrophoresis. The antibodies used in these techniques were radio-iodin-
ated so that the distribution of the protein could be detected.
One possible explanation for the uniformity of these antibodies is
that they are recovered from animals in which the magnitude of the
immune response is relatively feeble. It is possible that only a few cells
have been stimulated, and,
as a
consequence, the total antibody product
exhibits restricted heterogeneity. A corollary of this argument is that with
a heightened antibody response, there is a recruitment of a larger number
of cells and
as a
consequence the total antibody population is hetero-
geneous. Such
a
suggestion stems, in part at least, from the studies of
Little and Counts ( 1969).
A
homogeneous antigen DNP-lysyl-insulin
was synthesized and characterized, and, in this case, the attachment site
of
the DNP was regulated and not random. The antibody response to
this antigen was as heterogeneous as is usually the case when DNP is
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
43
coupled in a random fashion to other proteins. At present, there is no
satisfactory resolution of the dilemma presented by results with the
DNP-lysyl-papain an d th e DNP-lysyl-insulin, except to say th at th e
designs of the experiments differ and that the protein carriers are not
the same in each case. Such experimental variables may lead to opposing
data.
V.
Myeloma Proteins and Paraproteins with Antibody Activity
Since th e first observation 10 years ago tha t
a
human paraprote in had
rheumatoid factor activity (Kritzman et al., 1961) ,
a
growing number of
myeloma proteins and paraproteins from man and myeloma proteins
from BALB/c mice have been identified which react with
a
variety
of
antigenic determinants. These reactive proteins have recently been
reviewed by Metzger (1 96 9) . An extensive bibliography was cited an d
this need not be reproduced here.
Plasma cell tumors are now readily induced in BALB/c mice by
intraperitoneal injection of mineral oil or other suitable irritants ( Potter
and Boyce, 1962; Potter, 1968a). These tumors arise in the peritoneal
region 3-12 months after the oil injection. Th e majority pr od uc e IgA
immun oglobulins (Po tter, 1968b)-a finding which points to th e prefer-
ential occurrence of the neoplastic event in immunocytes in the vicinity
of the gastrointestinal tract.
The human myeloma proteins and Waldenstram macroglobulins have
activity for a variety of antigens, includin g 7-globulin, red cells in the
cold, streptolysin 0, heparin, lipoprotein, cardiolipin, and dinitrophenyl
ligands. Mouse myeloma proteins have been identified which react with
dinitrophenyl, th e C-polysaccharide of the pneumococcus, dextran, an d
several other substances. These findings have raised the exciting possi-
bility that the procurement of uniform antibodies is readily a t ha nd by
the simple expedient of screening with
a
panel
of
ligands
a
large number
of myeloma proteins for specific activity. In principle, at least, this
approach is a reasonable one, because m ost investigators would no w a gre e
that the myeloma proteins appear to be similar in all respects to normal
7-globulin, even though they are the products of malignant cells (Putnam
and Udin, 1953; Metnger, 19 69 ). Therefore, the un ique amino acid se-
qu en ce of th e antigen-binding site should be identical for both an induced
antibody and a myeloma protein if they both havc the samc specificity
and
if
both are drawn from the same subpopulation of y-globulin.
The myeloma proteins with antibody activity will not be discussed
in the same detail as was the case for the antibody responses described
above. This is because the theoretical and operational problems which
arise in t he
use
of these materials differ in many respects from
a
similar
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44 RICHARD
M.
KRAUSE
consideration of uniform antibodies stimulated by specific immunization.
For example, it is assumed, for all practical purposes, that the myeloma
proteins are homogeneous, and heterogeneity need not be as rigorously
excluded as is the case for antibodies stimulated by immunization. For
the myeloma proteins, it is only the question of antigenic specificity
which must
be
clarified. O n th e other ha nd , questions a bout th e specificity
of antibodies a re secondary, w here as the accumu lation of ev idence for
substantiating the uniformity of th e antibody is th e primary consideration.
O ne hum an IgG myeloma protein (Eisen e t al.,
1967)
and several
mouse IgA myeloma proteins which bind DNP ligands have now been
examined in great detail (E isen
e t
al., 1968; Sch ube rt
et al.,
1968).These
antibodies bind ligand selectively and binding is confined to the Fab
fragment. Such data an d fluorescence qu enc hing by bou nd Iigand parallel
the behavior of antibody which occurs after stimulation with DNP
protein conjugates, and, for these reasons, these myeloma proteins are
rega rded as antibody.
It
remains to b e clarified, how ever, if these pro teins
are in all respects identical to the antibody which occurs in response to
a specific antigenic stimuIus. In oth er words, a re these myeloma p roteins
really prototypes of the antibody to DNP which would have occurred
if
the animal or man had been immunized?
Tha
argument is speculative
and at the moment, perhaps, circular. The problem remains that these
proteins are, in fact, the produ cts of cells program med to m ake y-globulin
with a specificity which can only be determined at present by a screen-
ing procedure which uses a battery of antigens. If by fortuitous chance,
the myeloma protein reacts selectively with one antigen in such a
battery, this does not necessarily indicate that the myeloma protein is a
product of cells initially programmed to make antibody with this
specificity. This caution arises from several considerations which have
been reviewed by Eisen
et
al. (1 97 0) . Most impo rtant of these considera-
tions is the unexpected an d unexplained high incidence of myeloma p ro-
teins with D N P activity.
Over
350
mouse myeloma sera have been screened for antinitropheny l
activity (Eisen
et al.,
1968; Schuber t
e t al.,
1968), and approximately
6%of these were reactive. How ever, these proteins v aried enorm ously in
their affinity for ~-2 ,4-D NP -~-ly sine.ome reacted
so
weakly and in such
an anomolous way that they had lit t le or no resemblance to antinitro-
phenyl antibodies which are obtained by routine immunization. Eisen
et a2. (1970) have plotted the cumulative frequency with respect to
affinity for 6-DNP-lysine for fifteen mouse
IgA
myeloma proteins. These
ar e shown in Fig. 21. Only two of the proteins, 315 and 460, ha d mo derate
to high affinity for r-DNP-lysine. These two exhibited m ost of th e other
characteristics of conventional antinitrophenyl antibodies. This would
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SEARCH
FOR ANTIBODIES
WITH MOLECULAR
UNIFORMITY
45
0.10
:
.08
a
L
0
06
LL
W
I
\
K ( L / M ) - € - D N P- L - LYS I N E
FIG.
21.
Cumulative frequency for affinity ( K ) for binding e-DNP-L-lysine (at
4°C.) by some of the antinitrophenyl mouse myeloma proteins found in a population
of about
350
tumors.
(
From Eisen et
al.,
1970.)
indicate, if this trend continues, that about
1
have
an
affinity which
exceeds lo5
L I M
and are likely, therefore, to have combining sites, as
measured by affinity, analogous to conventional antibodies to nitro-
phenyls. But, as Eisen points out, this frequency of
1%
or even 0.01% s
still unexpectedly high if the transformation of plasma cells is a com-
pletely random process, and the nitrophenyls, as a group, are but one of
many thousands of non-cross-reactive antigenic determinants. One
possi-
bility for such a high incidence is that the activity is fortuitous and due
to nonspecific binding. Such a possibility, however, seems to have been
ruled out (Eisen
et
al.,
1970).
Antigenic cross-reactivity forms the basis for an alternative explana-
tion
for
the high frequency of mouse myelomas which bind DNP. It is
conceivable that an unknown antigen which cross-reacts with DNP has
forced a numerical prejudice from am ong the total imm unocyte pool in
favor of immunocytes that synthesize antibody against the unknown
antigen. As a consequence, at a subsequ ent t ime when the malignant
transformation occurs, a higher proportion of myelomas arise with cross-
reactive DNP activity than would have been anticipated by chance alone.
Several lines of evidence suggest this might be the case, but direct proof
is still lacking. It has been learned, for exam ple, th at m ouse m yeloma 315
reacts with naphthoquinones-a series of compounds wid esprea d in
nature which includes vitamin K. These types of compounds
a r e
coninion
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46 RICHARD M. KRAVSE
in the intestinal tract. Under such circumstances, it might be anticipated
that there would be an increase in the number of intestinal wall im-
munocytes which produce IgA specific for naphthoquinones. As a conse-
quence, when the malignant event occurs among the cells producing IgA,
a relatively high proportion of the tumors produce a myeloma protein
which is reactive with a naphthoquinone but is cross-reactive with a
DNP ligand.
The difficulty of identifying the precise specificity of a mouse myeloma
protein is well exemplified by a consideration of those that react with
pneumococcal C polysaccharide (Potter and Leon, 1968; Cohn et al.,
1969).
This is a complex polysaccharide which contains two polymers-
ribitol teichoic acid and a polymer of N-acetylgalactosamine phosphate
(Gotschlich and Liu, 1967; Brundish and Baddiley, 1968). Other com-
ponents of the C polysacchride include a diamino sugar, glucose, and
choline. Conventional antibodies, raised by immunization of rabbits with
pneumococci, commonly develop specificity for the N-acetylgalactos-
amine phosphate polymer of this complex carbohydrate. This does not
appear to be the case for the mouse myeloma proteins which react with
C carbohydrate. Leon has shown that these proteins appear to be specific
for the choline
of
the C carbohydrate (Leon, 1970). In an extension of
these studies, it has been observed that these myeloma proteins also
have specificity for phosphorylcholine and they also agglutinate red
blood cells coated with lecithin and lipoprotein (Leon, 1970). This
raises the possibility that these myeloma proteins are, in fact, analogous
to autoantibodies with specificity for cellular membrane determinants.
It should not go unnoticed that a number of the myeloma proteins, as
well as the Waldenstrom macroglobulins, have autoantibody activity.
Although the reason for this is not clear, there are several intriguing and
speculative possibilities. One possibility is that in each individual who
develops these proteins there are areas of focal inflammation with destruc-
tion of tissue and exposure of cellular antigenic sites. As immunocytes
accumulate and proliferate in these inflammed areas, it is possible that
a sizable proportion are converted to the production of antibodies against
a variety of “self” substances. Under such a circumstance, the selective
and unopposed proliferation of one of these cells would lead to synthesis
of a paraprotein or myeloma protein with autoantibody activity. Osser-
man and Takatsuki (1965) have stressed the possible importance of
chronic inflammation as the stimulus for the in situ occurrence of multiple
myeloma.
Potter ( 1970), more than anyone else, has systematically considered
the possibility that “the precursors of neoplastic plasma cells are actively
forming antibodies
to
immunogens that originate in micro-organisms that
inhabit the gastrointestinal tract.” He has conducted an extensive search
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SEARCH FOR ANTIBODIES WITH MOLECULAR UNIFORMITY
47
to
identify the polysaccharides of bacteria derived from the normal
enteric flora of the mouse which react with IgA mouse myeloma proteins.
Eighty-six IgA mouse myeloma proteins were tested for their ability
to
precipitate with forty-seven different antigens. Eight precipitated with
lipopolysaccharides derived from
Salmotielka, Escherichia
coli, Proteus,
and Pasteurella. Five precipitated bacterial antigens derived from the
normal intestinal flora isolated from BALB/c mice. An antigen was
extracted from a species of Ascaris which is an inhabitant of the mouse
intestinal tract. This antigen reacts with the same two myeloma proteins
that precipitate pneumococcus C polysaccharide.
If, in fact, antigens derived from en teric organisms are acting as
immunogens and stimulating the precursors of neoplastic IgA-producing
plasma cells, then su ch a view is subject to experimental test. It should b e
possible, for example, to raise BALB/c mice in the germfree state and
then selectively populate the bacterial flora with a single organism with
a known antigenic composition. This can conceivably lead to a pre-
dominant population of precursor immunocytes with
a
specificity for
antigens of this single bacterium. If th e hypothesis is correct and if the
myelomas have their origin in these percursor cells, a large proportion
of these myelomas should produce IgA with specificity for the bacterial
antigen. Such experiments ar c und er way (Potte r,
1970a).
An alternative app roac h for directing th e specificity of the plasma
cell
tumor has employed the
use of
antigens administered to the mouse
along with the mineral oil. These antigens have been injected eith er w ith
the oil that stimulates the tumor or
by
some other route. Taking a clue
from the apparent total commitment by nearly all plasma cells in the
rabbit to the synthrsis of antibody to streptococcal antigens following
intravenous administration of streptococcal vaccine, BALB/c mice have
been immunized intravenously for over a period of a year and have also
been injected intra periton eally a t intervals with m ineral oil. High levels
of precipitating antibody against the carbohydrate have been achieved,
bu t thus far i t is not certain tha t any of these antibodies ar e th e products
of plasma cell tumors (Eichmann, 1970). The final outcome of these and
other experiments along similar lines
is
awaited with interest. If the
experiments are successful, th e m eans w ould be at hand to procure, as in
conventional immunization, a large number
of
homogeneous immuno-
globulins which have specificity for defined antigens. Because there is
no obstacle to the successful transfer
of
a myeloma into a large number
of recipient mice, there would be no limit to the potential supply of any
one protein. But, most important of all, success in these experiments
would be
a
“demonstration that the prccursors of neoplastic plasma cells
are active
in
immunc rcsponscs” (Potter,
1970b).
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48 RICHARD
M.
KRAUSE
VI. Discuss ion and Summat ion
Are the means at hand to procure at will and in a reproducible and
predictable fashion, antibodies with molecular uniformity? All of the
work summarized here suggests that this is so. Antibodies to certain
antigens may, in fact, be much less heterogeneous than was formerly
supposed. For example, rabbit antibodies to bacterial polysaccharides
possess many of the features of the myeloma proteins which suggest
uniformity. Most important, in occasional rabbits, these antibodies occur
in concentrations between 30 and
60
mg./ml. With brisk responses such
as these, sufficient antibody can be obtained for extensive stru ctu ral work.
Although there
is
as yet inadequate proof for homogeneity based on
extensive amino acid sequence data, such work is progressing rapidly
in several laboratories, and all preliminary data suggest that selected
antibodies to microbial polysaccharides will possess a single primary
amino acid sequence and, therefore, in this last respect, these antibodies
will be comparable to the myeloma proteins.
At the time of the first publication which dealt with antibodies with
uniform properties in rabbits immunized with streptococci, it was sug-
gested that an examination of antisera from animals immunized with
other bacteria might reveal additional antibodies with properties indica-
tive of molecular uniformity (Osterland e t d. 966). From the present
advantage of hindsight, it now appears th at this prediction was a n und er-
statement. For example, intravenous immunization of rabbits with
pneumococci has yielded antibodies with uniform properties ( Haber ,
19 70 ). Preliminary results ( Gotschlich and Feizi, 1970) suggest that
intravenous immunization of rabbits with living Group A meningococci
yields antibodies with uniform properties to the capsular carbohydrate
which is a homopolymer
of
N-acetylmannosamine phosphate ( Gotschlich
et aZ., 1969).The list of possible bacteria that possess immunochemically
attractive carbohydrate antigens is almost endless. Among the many
choices, however, there are several which would merit attention in the
future. Streptococcus bovis, strain 19, possesses a dextran capsule (Kane
an d K arakawa, 19 69 ), an d it seems very likely, in view of the large bo dy
of information available on hum an antibodies to dextran th at an examina-
tion of the antibody response following immunization with these bacteria
would be especially rewarding.
Attention should also be called to the teichoic acid antigens of the
staphylococci. Torii et
al.
( 1964) have identified by immunocheniical
means,
01-
and ,8-N-acetylgalactosamine teichoic acid mixtures in antigen
preparations from staphylococci. Certain staphylococcal strains possess
a teichoic acid which is nearly all ,-linked, an d others possess a teicho ic
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SEARCH FOR ANTIBODIES
WITH
MOLECULAR
UNIFORMITY
49
acid which is nearly all P-linked. Immunization with these staphylococci
presents the interesting possibility of obtaining uniform antibodies to two
specific antigens which have a nearly identical chemical structure.
If
an
immunologist is prepared to scttle for antibodies to carbohydrates and
forego the advantagcs of antibodies to synthetic antigens, there is a
potentially large number of different bacterial antigens which can be
employed to generate antibodies with uniform properties.
At the moment there are three major requirements for success in
achieving high lcvels of uniform antibodies to bacterial carbohydrates.
The first is that the carbohydrate antigen must occupy the outermost
layer of the bacterial surface. In the case of the streptococci, this is not
a
natu ral occurrence. Only w hen th e protein antigens ar e digested away
with pepsin is the undcrlying carbohydrate exposed. The capsular
polysaccharides of the pneumococci are clearly on the periphery of the
cell, and, as a result, no special treatment of the vaccine
is
required.
Because the outermost carbohydrate antigens in some bacteria are
readily washed away during the preparation of the vaccine, it may be
necessary to use bacteria which are collected directly from the broth
culture and which have not been processed in any way. Gotschlich et al.
(1 96 9) , for example, found tha t only live meningococci, collected in th e
log phase of growth, still possessed the capsular carbohydrate. Late
growth phase cultures which had been killed and thoroughly washed
were nearly devoid of the antigen.
The second requirement for the successful stimulation of high levels
of uniform antibodies is the intravcnous immunization
of
a vaccine com-
posed of the whole bacteria. Alternative routes of immunization are less
effective. Use of the isolated carbohydrate alone is unsatisfactory. The
third requirement is related to the genetic background of the rabbit.
There is accumulating, although as yet only preliminary, evidence which
suggests that an immune response with 20 to
50
mg./ml. of precipitating
antibody is a trait which may be genetically transmitted (Braun et al.,
196 9). Any new research p rogram to procure rabb it antibodies with
uniform properties should employ a large number of rabbits from clearly
different stocks and breeds. From such
a
widely diverse g rou p of rabbits,
a limited number will have high responses. A portion of th ese high
responders will have antibodies with uniform properties.
If, indeed, rabbit antibodies with molecular uniformity are at hand,
to what purpose can they be employed? They should prove useful for at
least thre e different lines of investigation. These ar e th e structure-func-
tion relationship of antigens and antibodies and the topography of the
antigeii-combining site, the genetic control of the biosynthesis of the
immune globnlins, and the evolutionary mcchanisni responsible for their
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50
RICHARD M. KRAUSE
diversity. Finally, use of these antibodies in specially devised idiotypic
( a n d individual an tigenic specificity) experiments should afford a means
to monitor the em ergence of antibody populations over prolonged pe riods
of immunization.
It must be left for a later review to cope with what will undoubtedly
b e
a
massive accumulation of sequence data on antibodies with uniform
properties such as those described here. If tw o antibodies a re draw n from
the same subclass of IgG and possess the same class of light chains and
identica l allotypic markers a nd if each is specific for a distinct but related
antigenic determinant, their specific amino acid sequence should be of
value to describe the unique topography of the antibody-binding site.
In this connection, rabbit antibodies with restricted heterogeneity may
have special application in affinity labeling experiments which have
been designed t o locate the antigen -bind ing site. Affinity labeling reagents
bind specifically
as
hapten to the site, but, in addition, bind irreversibly
to an amino acid in the site. It is then possible to identify the peptides
of the heavy and light chains to which the reagent is attached. This
approach to search out the antigen-binding site has been described in
detail and will no t be reviewed here (Singer and Doolitt le, 1 96 6). T h e
subject is mentioned, however, because it is conceivable that the rabbit
antibodies to streptococcal carbohydrates and pneumococcal polysac-
charides will be useful proteins for affinity labeling studies. T h e tec hn iqu e
has been employed, for example, for equine anti-P-lactoside antibodies
( Wofsy
et al.,
1967a) and for rabbit antisaccharide antibodies
(
Wofsy
et al., 196713). T he one obvious advan tage
of
the antibodies to the bac-
terial polysaccharide for such affinity labeling studies is their occurrence
in high concentration. This assures an ample supply of antibody for
extensive investigative work.
Progress on the structure and amino acid sequence of human and
mouse 7-globulins has proceeded rapidly because th e occurrence
of
multi-
ple myeloma in these species assured a ready source of homogeneous
proteins. Now that homogeneous rabbit antibodies are available, many
questions can be examined for this species as well. It has been already
learned, for example, that a definite homology exists in the variable
region between the human K and rabbit l ight chains. When the amino
acid sequences of rabbit antibody light chains (b4, and, therefore, K )
are aligned against their human
K
counterparts, a definite homology is
suggested between the N-terminus of the human and the rabbit variable
regions. Such an alignment is depicted in Table IV. Since a similar
homology has been noted between the common C-terminal peptides
(Do olitt le and Astrin, 1967; Hood et al., 19 67 ), further sup por t is gained
for the hypothesis that rabbit
K and
human I( light chains descended from
common ancestral genes in the variable
as
well as the common region.
Furthermore, because of the addition of an extra N-terminal alanine in
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SEARCH F O R ANTIBODIES WITH MOLECULAR UNIFORMITY
51
some rabbits (i.e., R27-11) and because of the deletion of a N-terminal
residue in other rabbit light chains (i.e., 1324-61 slow Component), it is
apparent why an N-terminal analysis of pooled rabbit light chains failed
to reveal significant homology with human
K
chains (Doo li t tle an d
Astrin, 1967).
Although the N-terminal sequence data on the light chains of strepto-
coccal antibodies are still fragmentary, several observations merit
comment,
A comparison of the am ino acid alternatives at t h e first thre e N-termi-
nal positions of the light chains of isolated antibodies and the light chains
of preinimune y-globulin from the same rabbit indicate that immuniza-
tion may select a relatively uncommon species from among the many
alternatives of normal y-globulin. For example, isoleucine accounts for
less than 6% f t h e amino acid residues recovered from th e first N-terminal
position of a single rabbit’s preimmune light chains. And yet, in the
Group C antibody recovered from this rabbit after immunization, iso-
leucine was the major N-terminal amino acid of the light chains (Hood
et al., 1969). Furthermore, a similar result has been observed with a
surprising frequency for a number of streptococcal antibodies isolated
in this way (Hood
et al., 1970).
Perhaps more striking is the fact that
light chains with predominantly N-terminal isoleucine have been iso-
lated from both Group A and Group C antibodies. This suggests that
two antibodies, each with a distinct and different immunological speci-
ficity, have been drawn from the same infrequent subpopulation of
y-globulin. N-terminal se qu en ce amino acid analysis on th e light chains
of 9 rabbit antibodies indicates
a
division into at least
3
variable region
subgroups (Hood
et
al., 1970). Furthermore, species specific residues are
observed which suggest that rapid gene expansion has occurred since
man and rabbit diverged.
In the earlier sections of this review, it was emphasized that at least
two distinct antibody compogents can be isolated from a number of
hyperimmune rabbit streptococcal antisera, such as example d in Fig. 17.
Each of the two coniponents in such antisera possesses unrelated indi-
vidual antigenic specificity. T he light chains of e ach c om pon ent may
have a distinct N-terminal amino acid sequence. This is illustrated by the
data in Table IV on two antibody components isolated by preparation
electrophoresis from G rou p
C
antiserum R24-61. Alanine is the N-terminal
amino acid of the light chains of the slow component, whereas isoleucine
is the corresponding residue of the fast component. In this rabbit, the
two antibodies appear to have been drawn from different variability
region subgroups.
Because rabbit antibodies which have molecular uniformity are pre-
dominantly uniform with respect to all of the allotypic loci thus far
examined, they should
be
useful proteins for immunogenetic studies. It
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52 RICHARD
M.
KRAUSE
should be possible, for example, to clarify the ambiguity which still
surrounds the molecular determinants of certain of
the
allotypic specific-
ities, and
it
should
be
possible to define in
a
very precise way th e location
of these molecular determinants on the H a nd L chains. Such informa-
tion may eventually provide a rational basis for selecting among the
various mechanisms of genetic control postulated for antibody synthesis.
It remains finally to comment on recent studies which deal with the
idiotypy or individual antigenic specificity of rabbit antibodies to bac-
terial polysaccharides. Streptococcal antibodies produced by a rabbit
during repeated courses of immunization may elicit either identical or
distinct individual antigenic specificities ( Eichmann et al. , 1970 b). Firs t
and second immunization antibodies from a single rabbit which have
identical individual antigenic specificities have an identical electro-
phoretic mobility. Furthermore, the same antiserum may possess two
electrophoretically distinct antibody components, each one possessing
its own distinct individual antigenic specificity. These findings are in
agreement with the recent studies of Nisonoff
e t
al. (1970) and by O udin
and Michel ( 196 9a,b ). Furthermore, Ou din an d M ichel ( 1969a,b) and
Braun an d Krause (1968) observed that a rabb it may produce antibodies
with two or more idiotypic specificities when it is immunized with one
or more antigens.
T he recurrence d uri ng second immu nization of antibodies with an
individual antigenic specificity identical to that
of
antibodies produced
during primary immunization suggests that the same cell population,
present during primary immunization, has re-emerged with reimmuniza-
tion and,
as
a consequence, synthesis of identical antibody molecules
occurs in both instances. In view of the fact that an antibody-producing
cell does not survive longer than a few days, recall of antibodies with
specificity similar t o that present during the first immunization reinforces
the suggestion that cells with
a
memory function must be involved in
the recognition of the antigen and subsequent antibody synthesis.
There is no satisfactory explanation for the occasional occurrence of
a
high concentration
of
homogeneous antibodies in rabbits following
immunization with streptococcal or pneumococcal vaccines. One possi-
bility is that a cell population with numerical superiority over all others
is present before immunization and is stimulated by it. All
of
the indi-
vidual antigenic specificity data and light-chain sequence data, however,
clearly indicate that this is not the case. Instead it appears tha t immuniza-
tion may stimulate a minor rather than a
major cell population. Further-
more, the light chains of the antibody product
of
these cells have
an
N-terminal sequence which is a representation of
a
minor species of the
normal serum 7-globulin. It would seem that the use of individual anti-
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SEARCH F O R ANTIBODIES WITH MOLECULAR UNIFORMITY
53
genic specificity or idiotypy and an elucidation of the amino acid
sequence of specific antibody light chains will facilitate an examination
of
the cellular events that lead to
the
imniune response.
ACKNOWLEDGMENTS
The author is indebted to Dr. Maclyn McCarty,
Dr.
Leroy Hood, Dr. Charles
Todd, Dr. Thomas Kindt, and Dr. Klaus Eichmann for their critical review
of
the
manuscript. The final text has drawn heaviIy from their contributions and suggestions.
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St ruc tu re an d Func t ion of YMM a c r o g l o b u l i n s
I
.
I1
I11.
IV.
V
.
V I
.
VII
.
VIII
.
IX
.
X .
X I .
H
ENRY
METZG
ER
Art hrit i s and Rheumatism Branch. Notion al Institute o f Arthrit is and Metabolic Diseases.
National Institute
o f
Health. Bethesda. Maryland
Introduction
. . . . . . . . . .
Isolation and Storage of Macroglobulins
A
.
Ge nera l Me thod s of Isolation . . . . .
B. Specific Methods of Isolation . . . . .
C
.
Storage . . . . . . . . . .
Structure of Mammalian Macroglobulins
. . . .
A. Physical Properties . . . . . . .
. . . .
.
. . . . . . .
Chemical Properties
Subunits . Polypeptide Chains. and Proteolytic Fragments
A. Reductive Subunits . . . . . . .
C
.
Proteolytic Fragments of yM Macroglobulin
. .
Low
Molecular Weight Macroglobulin-Like Proteins
.
B . Polypept ide Chains . . . . . . .
Functional Properties
of
Macroglobulins . . . .
A. Interaction with Antigens
. . . . . .
B.
Interact ion wi th the Complement Sys tem . . .
C
.
Interactions with Other Proteins
. . . .
D. Interactions with Cells
. . . . . . .
Genetic Basis of Macroglobul in s t ructure . . .
A. Evidence for Subclasses . . . . . .
B
.
Allelic Markers
. . . . . . . .
C . Idiotypic Markers . . . . . . .
Biosynthesis and Metabolism of Macroglobulins
. .
A
.
Biosynthesis
. . . . . . . . .
B
.
Distribution . . . . . . . . .
C . Rates of Synthesis and Catabolism . . . .
Macroglobulin-Like Proteins from Nonniammalian Species
Role of Macroglobulins in the Immune Response
.
.
A
.
MacrogIobulins As Antigen Receptors
. . .
B. Macroglobulins in the Control of Antibody Synthesis
Prospects . . . . . . . . . .
References . . . . . . . . . .
. . . 57
. . .
59
. . .
59
. . . 60
. . . 60
. . . 60
. . .
60
. . .
71
. . . 73
. . .
73
. . .
76
. . . 85
. . .
88
. . . 89
. . .
89
. . . 94
. . .
97
. . . 97
. . . 98
. . . 98
. . . 99
. . . 99
. . .
100
. . .
00
. . .
01
. . . 01
. . . 02
. . . 104
. . .
105
. . .
06
. . . 106
. . .
108
I
.
I n t r oduc t i on
To classify rigorously a protein one must ultimately be able to refer
to th e gene
(or.
if th e prote in
is
composed
of
several polypep tide chains.
the set of genes) which codes for the primary structure of the protein .
57
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58 HENRY METZGER
Specific polynucleotide sequences may in certain instances become avail-
able (Shapiro et
d.
96 9) , bu t for some time to com e it will be necessary
to define genes in terms of amino acid seqnences or genetic (antigenic)
markers or both. Such data are largely unavailable for the yM immuno-
globulins. These proteins can be defined, therefore, on the basis of
derivative properties only. Deciding which of these properties must be
present to accept
a
protein as
a
member of this class is perforce arbitrary
and classification must, therefore, be considered provisional.
Historically yM immunoglobulins were first recognized in the sera of
cattle an d horses (H eidelb erge r and Pederson, 1937; Kabat, 1 961 ). It
was found that in addition to those antibodies having
a
molecular weight
of
150,000
to 160,000 an d
a
y
mobility on electrophoresis,
a
class of anti-
bodies having a molecular weigh t of around 1,000,000 an d a somewhat
faster electrophoretic mobility than the former group, was often elicited.
These macroglobulins had a higher carbohydrate content than the low
molecular weight y-globulins and were readily dissociated into subunits
upon exposure to reducing reagents. Rabbit antisera were elicited which,
after appropriate absorption, were specific for these proteins. Hence-
forth, immunoglobulins which cross-reacted strongly with such antisera
an d/ or h ad physicochemical properties similar to th e pro teins originally
described, were considered yM immunoglobulins.
Most of this later work was performed with mammalian proteins,
mostly from humans, and it is only for proteins from inaminalian species
that
the term yM immunoglobulin can
be
used with assurance. Although
it is likely that many high molecular weight immunoglobulins from more
primitively arising species are evolutionarily homologous to mammalian
yM, it seems reasonable to aw ait furth er sequence d ata before attempting
a
final classification. For this reason I have segregated the discussion on
mammalian yM proteins from that on nonmammalian yM-like proteins.
This more accurately reflects the state of our knowledgc.
Many Waldenstroin macroglobulins have been examined with a view
to understanding their structure and their relationship to y M proteins
which are normally present. As with the analogous myeloma proteins,
such macroglobulins are obtainable in large amounts, and, being homo-
geneous, are amenable to detailed chemical and genetical analysis.
That some of them have well-defined combining activity (reviewed in
Metzger, 1969a) makes them particularly attractive to study. It is, of
course, true that, arising as they do in t h e course of a neoplastic process,
these proteins could in certain instances be sports. Nevertheless, ex-
perience has shown that extrapolation of findings on such proteins to
the structure
of
normally arising immunogIobulins is usually valid.
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STRUCTURE AND FUNCTION OF
y M
MACROGLOBULINS
59
1 1
Isolation and Stcrage of Macroglobulins
A.
GENERAL ETHODS
F
ISOLATION
Th e yM macroglobulins a re commonly isolated by following an initial
selective precipitation with fractionations based primarily on molecular
size (e.g., gel filtration) or on a combination of size and charge (e.g.,
zone electrophoresis).
Many macroglobulins are euglobulins and are readily precipitated
from serum by dilution w ith distil led w ater (D eu tsch and M orton, 19 58).
Curves relating yM solubility to ionic strength have been published by
Mandeina et al. (19 55) and M art in (19 60) , but , of course, variations
may
be
expected depending on the specific protein being studied, the
solvent pH, and temperature. Schultze and Heremans list a variety
of
solvents from which yM pro teins will precipitate (s ee Tab le 42, Schultze
and Heremans, 1966).
When contamination with low-density lipoproteins is
a
problem,
raising the density of the solution and floating off the lipoproteins
through centrifugation has led to effective delipidation ( Chaplin
e t
al.,
1965; Inm an and Hazen, 196 8). Lipoproteins ma y also be precipitated
with 0.5%dextran sulfate (m ol. wt.
=
560,000) in the presence of 0.09 M
CaCl, (Burstein and Samaille, 195s)
as
has been described by Chesebro
and Svehag
(1969)
for the purification of rabbit yM. Precipitation at
neutral pH with sodium phosphotungstate in the presence of MnCl,
has also been used ( Burstein, 1963).
Fractionation b y size is performed by gel filtration on Sep hadex G-200
( cross-linked dextran ), Bio-Gel
P-200
(
cross-linked polyacrylamide ),
Sepharose ( agarose beads), or related materials. For smaller quantities,
ultracentrifugation is useful ( Kunkel et al., 1961; Stanworth, 1967 ). W ith
adequa tely large columns, macroglobulins can b e separated from
th e yM macroglobulins (see, e g ., Chesebro a nd Svehag, 1x9). imilarly,
if it is undesirable to have high molecular weight yM polymers present
(
Section III,A,I ), they can be eliminated by successive gel filtration
(F. Miller and Metzger, 1965a; Morris and Inman, 1968).
Fractionation on th e basis of charge can be affected b y diethylam ino-
ethyl cellulose ion-exchange chromatography ( Chaplin
e t al.,
1965;
Fahey and Terry,
1967)
or zone electrophoresis on starch, Pevikon
( Muller-Eberhard, 19 60 ), or agar (O no ue e t al., 196 7). Conservative
pooling of thc ion-exchange effluent can also help to eliminatc traces of
,&lipoprotein ( Chaplin
et
d. 965) .
Excellent yields of purified rat yM macroglobulin have been re-
ported
b y
Fisher and Canning (1966) using sucrose density gradients
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60
HENRY METZGER
in the BIV zonal centrifuge. This method allows for large batch process-
ing and may be particularly useful for the isolation of y M from small
animal sources.
B. SPECIFIC
METHODS
F
ISOLATION
Advantage can be taken of a known binding activity to isolate the
y M antibody. The antigen-antibody complex is washed, dissociated, and
the antibody and antigen separated. The latter step is, of course, most
easily accomplished if the antigen is bound to an insoluble matrix. Dis-
sociation at acid pH's is most often used but low molarities of urea or
KSCN (Dandliker et al., 1967; Stone and Metzger, 1969) can also be
used and may be safer. Where complex formation is promoted by low
temperatures ( cold agglutinins, mixed cryoglobulins
,
simple warming
may be an effective adjunct. In general it can be stated that native
macroglobulins are fairly hardy molecules (Putnam, 1959) and will
withstand low pH's e.g., 0.1 N acetic acid (Robbins e t al., 1967) and
elevated temperatures ( <60°C.) for limited time periods (Pike, 1967;
Murray
et
al.,
1965a).
C.
STORAGE
A
study on normal pooled sera conducted under the auspices of the
W. H.
0.
International Reference Centre for Immunoglobulins (Rowe
et al., 1970) showed that storage of either frozen or freeze-dried serum
at
-20°C.
was adequate. Criteria used for stability were repetitive,
single, radial diffusion assays and gel filtration. In our experience with
two Waldenstroni macroglobulins which show binding activity to human
yG
( Metzger, 1967) and nitrophenyl derivatives (Ashman and Metzger,
1969), respectively, storage at -20°C. has been satisfactory. Storage of
isolated macroglobulins in borate-NaC1 buffers (pH 8.0) at 4°C. has
also not led to detectable changes in these preparations over many
months.
Ill.
Structure
o f
Mammalian Macroglobulins
A.
PHYSICALROPERTIES
1. Sedimentation Rate
A macroglobulin preparation isolated by one of the above-mentioned
methods will usually be heterodisperse when examined by analytical
ultracentrifugation. The major component-comprising about 85%of the
protein-will have a corrected sedimentation rate of 18
S
to 19
S,
where-
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61
TRUCTURE AND FUNCTION O F yM MACROGLOBULINS
as
minor components of
2 2 9 s
a nd
3 8 s
make up about
15
a n d
5%of
the total protein, respectively ( Deutsch and Morton, 1958; Miiller-Eber-
hard and Kunkel,
1959) .
A
very wide range of sedimentation constants for each of the com-
ponents has been reported in studies of large numbers of macroglobulin-
emic sera (as opposed to isolated macroglobulins). For example, Ratcliff
et al. (1963) reported an average sedimentation constant of 19.9 S bu t
a range of 14.2 S to 22.2 S for th e major component. Correspo nding values
were
29.5 S (20.1 4 S-34.3
S
)
a n d
37.6 S (24 .8S 3 9 . 0
S ) for the heavier
constituents.
A
statistical analysis of forty-two sera by Filitti-Wurmser
et al. (1964) indicated a bimodal distribution-about 60%of the major
components having a mean sedimentation constant of
17.0
( r a nge
15.6-
17.9) a nd
40%
having a mean sedimentation constant of 18.3 ( r a nge
17.3-19.3). The sedimentation constant of the major component and
the intermediate one appeared related. Thus, in those sera in which
the major component had a mean ~ , 9 ? , ~f 17.0 the intermediate com-
ponent had a mean s, ,,, of
23.9;
for the second group the corresponding
value was 27.0. Only three of th e forty-two specimens failed to show
such a correlation.
It is di5cult to evaluate these data. They involve several correction
factors and it would be helpful to have some analyses on the purified
macroglobulins from such sera to verify t h e ad eq ua cy of t he analyses.
Th e concentration d epen den ce of sedimentation app ears to be
highly variable even on purified preparations. In the equation
s o (I - kC), here C
is
in grams per cent, k in thr ee recent studies can be
calculated to be 0.098 (Suzuki and Deutsch, 1966), 0.257 (F. Miller
and Metzger, 1965a), and 0.648 (Morr is and Inman, 1968). It seems
possible that the unusual shap e of the molecule (se e be low ) may in-
fluence the concentration dependence and extrapolated sedimentation
constant under relatively minor experimental variations.
Reports on the dissociability of components heavier than 19 S are
contradictory. Franklin (
1960)
found almost complete dissociation of
human 29
S
components to 19 S at p H 3.5, and Lamm and Small (1966)
found a halving of the molecular weight
of
rabbit 2 9 s component
(1 .8
x lo6 to
9
x l o 5 ) in
5
M guanidine. Reversible dissociation of both
2 8 s a nd 3 2 s to 1 9 s components was observed at alkaline p H a nd
elevated temperatures by Suzuki and Deutsch (1966) . On the other
hand, dissociation was not observed by others under conditions where
cleavage of covalent bonds would not be expected (Kunkel, 1960; Rat-
cliff et al., 1963) . In the th ree studies mentioned above, disulfide cleavage
due to trace amounts of reducing reagents or due to protein-bound sulf-
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62
HENRY
METZGER
hydryls was not rigorously excluded so that this remains as a possible
explanation for some of the disparate results. Nevertheless, it is clearly
possible that both noncovalent and covalent polymerization
of
t he
18
S-
19S component might occur as it does with oth er proteins.
The
29 S a nd 35 S components are evident in unprocessed sera and
the 19
S
com ponen t can be freed of them by gel filtration (Se ctio n 11,A).
Thus,
although the heavy components may arise from "rough" handling
of the yM monomer, they do not appear to be due to that alone nor to
an equilibrium aggregation phenomenon.
Suzuki and Deutsch ( 1966) have investigated three macroglobulin
preparations which contained an interesting 22
S
component in addi-
t ion to the
19s
and 2 8 s components .
The
22s component was con-
verted to the 1 9 s species at both acid and alkaIine pH and at elevated
temperatures. Evidence was given that this was not a yM anti-yM anti-
gen-antibody complex. T he authors suggest tha t th e 2 2 s species is a
noncovalent dimer of the 1 9S , bu t a change in the sha pe of the 19s
component was not excluded. Molecular weight studies
on
the 22S-rich
preparations could easily differentiate between these alternatives.
2.
Diffusion Coeficient
A
limited number of diffusion analyses
on
macroglobulins have been
reported. Kabat 's data (1939) on a horse antibody preparation showed a
somewhat erratic concentration dependence and yielded a value of
1.8x
lo-?
cm.*/second. Values for cow and pig macroglobulins were
1.64 and 1.69
x
respectively. The data of F. Miller and Metzger
(1965a) on a Waldenstrom macroglobulin showed a linear negative
concentration dependence, the extrapolated value being 1.75 x lo-'.
Suzuki and Deutsch (1967) reported
a
value of 1.71
x
lo-', an d Beale
and Buttress (1969) gave a value of 1.73 x l@' cm.*/second fo r a 0.39
protein solution.
A
relatively high value, 2.22
x
low7
cm.2/second was
reported by Fisher an d Canning (1966) fo r ra t macroglobulin.
T h e diffusion an d sedimentation coefficients can b e used to calculate
a frictional ratio f / f o ) , a number related to the hydrodynamic asym-
metry of the molecule. Values close to 2 have been commonly obtained
for yM (Kabat, 1961; F. Miller and Metzger, 1965a).
3. Intrinsic Viscosity
A
broad range
of
intrinsic viscosity values have been reported for
yM: from 0.06 deciliter/gm. (Kovacs and Dau ne, 1961) to 0.30 deciliter/
gm. (M artin, 19 60). Ca refu l studies by Jahnke
et
al. (1958) on four yM
prep aration s gave a rang e of 0.106 to 0.153 dec iliterlgm.
A
highly purified
preparation gave 0.162 deciliter/gin. in our own work (F. Miller and
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STRUCTURE AND FUNCTION
OF -yM
MACROGLOBULINS
63
Metzger, 1965a). No change was observed when the ionic strength was
raised appreciably so that charge effccts were excluded in the latter
study.
4 . Partial Specific Volume
Kabat
(1939)
measured the partial specific volume of horse anti-
body and obtained 0.715 cm.’/gm. F. Mil ler and Metzger (19654 ob-
tained 0.723 & 0.001 c ~ n . ~ / g r n .or a human Waldenstrom macroglobulin,
and a value of 0.717 cin.?/gin. has bee n calculated (C h e n
et al.,
1969)
from the amino acid and carbohydrate compositions recorded by Heim-
burger et al. (1964) . A value of 0.730 cm .3 /gni . was similarly calcula ted
for rat macroglobulin
(
Fisher and Canning, 1966).
5.
hlolecular Weight
A
wide rang e of m olecular wcights has been reported for y M immuno-
globulins. Several of the earlier results arc listed in Kab at (1 96 1). Most
of the recent studies have yielded molecular weights between 850,000
and 1,000,000 for human Waldenstrom macroglobulins ( F. Miller and
Metzger, 1965a,b; Suzuki and Deutsch, 1967; Chen et al., 1969; Beale
and Buttress,
1969). A
much wider range, 620,000-1,180,000, has been
described for hu ma n norm al an d Waldenstrom macroglobulins by Filit t i-
Wurmser and Hartmann (1968 a) . Rat y M was calculated to have a
molecular weight
of
770,000 (Fish er and Canning, 1966 ), an d rab bit
yM was reported to have a molecular weight of 900,000 by Lamm and
Small (1 96 6). Th e “best” value (of 990,000) for the molecular weight of
cow, horse, and pig macroglobulin antibody
(
Kabat, 1939) probably
needs to be corrected do wnward by abo ut lo%, since the sedimentation
values on which these results were based could not at that time be
adequately corrected for temperature variations occurring during the
ultracentrifugal analysis ( Shulmnn, 1953)
.
It is not possible to evaluate these disparate results in the absence
of
other data relating to the number, size, and yield of the subunits,
polypeptide chains, disulfide bonds, and protcolytic fragments, as well
as to the numbcr
of
antigen-combining sites an d t he electron-microscopic
pictures.
F.
Ultraviobt Absorption and Color Coeficient
Several values for the ultraviolet absorbancy of yM proteins have
been published. Those
I
am aware of are given in Table I along with
some unpublishcd data. In the absence of direct data, to assume an
extinction cocfficicnt of 1.25 wou ld seem to be safest. Data on non-
mammalian protcins a r c given in Section
IX.
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64 H E N R Y METZGER
TABLE I
E X T I N C T I O N COEFkTCIENTS
FOR HUMAN
ND
RABBIT
hf
Protein
Method
usedc
. 1%
Specimen ReferenceD Type" %go mr(l a m . )
~~ ~
Y M W W
yMWng (submit)
Y M W n s
YAfL,,
Y M M ~ ~
Y A f 0 d
(TMtOd
Human
yM
Human ybl
Human y h I
Rabbit yM
Rabbit
yM
(anti-polysaccharide)
(ant i-benzenearsoriate)
(ant i-benzeneat sonat e)
( 3 )
(7)
- 1.22
W 1 .35
N I .33
- 1.34
-
1.32 zk
0 . 2
(1.21)
D.W.
D.W.
R.I.
R.I.
R.I.
R..I.
R.I. ,Ke
D.W.
Kf
:
References:
(1)
F.
Miller atid Metzger
(1965a).
(2)
Metzger (1969b).
( 3 ) Merler et d. 1968).
(4)
Mihaesco
(1967).
* W = Walderistrom macroglobulin; N = normal pooled niacroglobulins. Where
light-chain type is known it is indicated.
c
D . W .
= based on dry weight,;
R.I .
= based on refractive index increment, in
0.15
N
NaCl. Refractive index incrementa assumed
to
be 0.00188 (Kabat.,
1961);
K
=
Kjeldahl or equivalent,.
d The prot,eiri being sequenced by Wikler el al. (1969); see
also
A.
P. Kaplan and
Metzger
(1969).
e I r i
this study t,he refractjive index increment was asslimed
to
he
0.0019
and the
nit,rogeii cotitmerit5%. Agreement was said to he -47,.
f
The value
of 1.34
was based
on an
assimied nitrogeri of
16y0.
If the true nitrogen is
-14.5(;', (see Section III,B,I) then the ext,inctiori Coefficient is 1.21.
y
Nitrogen cont~entlwas assumed to he 14.5%.
The good agreement between the extinction coefficient for yMwas
determined 011 the basis of dry weight and on the basis of an assumed
refractive index increment of 0.00188 (green l ight ) (Kab at , 1961; Table
I ) suggests that t he latter value is app rop riate for macroglobulins.
An
extinction coefficient
at
700
nip,
for
a
yM
macroglobulin assayed
by th e Folin-Ciocalteu phenol method ( Kabat, 1961) has been p ublished
(F.
Miller and Metzger, 1965a). Over the Ihiear part of the curve, an
absorbaiicy
of 24.5
for a
0.1%
d ry w eigh t) solution was recorded. Values
for huiiian
yG
and hu m an serum albumin were 23.2 a nd 15.4, respectively,
in tha t same study.
5 )
Schultze and Heide, unpublished observation
cited in Schult,ze and Heremans (1966).
(6) Onoue et at. (1965).
(7)
Hoyer
el aE. (1968).
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STRUCTURE AND FUNCTION
OF
yM MACROGLOBULINS
65
7 .
Electrophoretic Mobility and Isoelectric Point
The isoelectric point of yM macroglobulins varies widely from
protein to protein: 5.1-7.8 in one early stu dy (D eu tsc h an d Morton,
1958). Though of historical interest in defining levels of heterogeneity
among immunoglobulins, such data do not yield useful insights into
molecular structure. The variation in isoelectric points is only partially
reflected in zone electrophoretic analyses, such as immunoelectrophoresis,
since
the
hydrodynamic properties of the molecules restrict their mobil-
ity. Still, on preparative runs a relatively wide range of mobilities is
observed ( Kunkel, 1960).
8. Refractive Properties
Optically active side-chain absorption bands complicate the in-
terpretation of optical rotatory dispersion ( O R D ) and circular dichroism
( C D ) s pec tra
of
proteins in terms of polypeptide backbone conforma-
tion. Although the ORD spectra from yG and yM molecules differ
(Jirgensons, 1960; Dorrington and Tanford, 19 68), there are common
features which are of interest. W he n th e data ab ove 300 mp. ar e analyzed
according to the equation of M offitt an d Yang (1 95 6) , th e values for
a,
ar e from -163 to -206 for yM an d som ewhat lower (-290 to -320)
for yG proteins. Correspo nding valucs for b , are 0-20 for both yM an d yG
(Callaghan and Mart in, 1964; Dorrington and Tanford, 1968; Wet te r
et al., 19 66 ). Although these values speak against extensive h elical re-
gions, considerable @ -structuremay be present as in -yG immunoglobulins
(Ashman et al., 19 70 ). Th e marked changes induced by extremes of
p H a n d high m olarities of urea also suggest considerable tertiary
structure in these proteins. A remarkable finding is that the latter
folding appears to
be
divided into noninteracting domains. That is, the
ORD spectrum appears to be the simple algebraic sum of the spectra
for the fragments released by proteolysis of both yG and yM immuno-
globulins and for the subunits released by reduction
of
the 19S yM
molecules ( L . A. Steiner and Lowey, 1966; Dorrington and Tanford,
196 8). Fur ther details on the O R D an d C D spectra of yM proteins can
be found in Dorrington and T anford (19 68) and in Ashman
e t ul.
(1970) .
9. Fluorescence Polarization of Macroglobulin-D ye Conjugates
The high intrinsic viscosity (Section III,A,3) and high frictional
co-
efficient (Section III,A,2) of yM are consistent with macroglobulins be-
ing asymmetric, rigid ellipsoids. However, several other properties of
these molccules are not consistent with such a picture: ( a ) the lack of
interaction between the subunits once the single intersubunit disulfides
are broken (Section IV,B,S);
( b )
he marke d susceptibility to proteolytic
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66 HENRY METZGER
digestion leading to the formation of fragments that show no tendency
for noncovalent interactions (Section IV,C,2) ; a nd (
c )
the independence
of the ORD “domains” referred to above (Section III,A,8). All these
latter findings suggest that the yM pentamer may have considerable
flexibility and that this rather than asymmetry accounts for the hydro-
dynam ic properties.
By stud yin g th e variation in fluorescence pola rization of YM-dye con-
jugates w ith solvent viscosity, it should b e possible to obtain experimental
verification for such a flexible model (Weber, 1953; R. F. Steiner and
Edelhoch, 1962).
Two studies that utilized this technique have been published in ad-
equate detail to evaluate (Metzger et
d.
966a; Knopp and Weber,
1969). Metzger et al. ( 1966a) studied l-dimethylaminonapthalene 5-
sulfonyl chloride (DNS) conjugates of
yM,
the subunits (Section IV,A)
and t he tryptic Fa bp fragments (Section IV ,C ). Th e relaxation times
were respectively 80t , 69, and 58 nscc. Since even
a
rigid sphere of
ap pro pri ate mass should have given a relaxation time of ab ou t 730 nsec.
for
yM,
they interpreted their data as indicating significant internal
rotations in the nanosecond range. The relaxation times were independent
of th e degree of conjugation, an d several experiments (P er lm an an d
Edelho ch, 19 67) failed to give evidence for rotation of th e d ye molecules
independently of the protein.
Knopp and Weber (1969) investigated pyrene butyric acid con-
jugates of human
y M .
These conjugates have fluorescent lifetimes of 100
nsec.-about 8 times longer than the
DNS
conjugates-and are, there-
fore, mo re app rop riate for picking u p lon ger relaxation times. They
obtained a value of
1000
zk 200 nsec. for the
yM
pentamer and about 200
nsec. for the reduced protein.
A t
low values of TI7 their plots showed
considerable deviation from a straight line, indicating the presence of
shorter relaxation times. Knopp and Weber interpret their data as in-
dicating that the molecule is fairly symmetrical but do not comment on
the implied discrepancy, therefore, with the hydrodynamic data. Both
sets of investigators agree that there must be some flexibility to the
molecule but the exact time constants and extent of such movements
for the molecule as a whole and for the individual subunits and Fab
regions remain uncertain. A recent paper by Yguerabide et
at.
(1970)
reporting the use of nanosecond fluorescence polarization with yG anti-
bodies directed to the fluorescent chromophore is promising in this
respect. They were able to estimate separately the rotational correla-
tion times of the F ab fragments and th e whole yG as well as the angular
range of Fab motion. Comparable studies on
yM
antibodies would be of
obvious interest.
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STRUCTURE
AND
FUNCTI ON
OF YM MACROGLOBULIXS
67
10. Electron Microscopic T rltriistructure
An extensive review of ultrastructural studies on Y G and yM immuno-
globulins was published by Green (1970) in the
previous
volume of this
series. Briefly, thc elegant studies by Svehag and
his
colleagues on
Clialdenstrom. normal human and rabbit macroglobulins
(
Svehag e t ul.,
FIG.1.
Electron micrograph of
a
single
mouse IgM
selected from
a
micrograph
by
Parkhouse et al. (1970). Details of preparation are given in Parkhouse et al.
(1970).
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68 HENRY METZGER
1967a,b; Chesebro e t aZ. 1968) first gave direc t visualization of th e flexi-
ble circular pentamer predicted from physicochemical investigations.
Study of the papain
F(
c ) ~
and peptic
F(
ab’)2
fragments (Section
IV,C) (Svehag e t
al.,
1969) added further details.
It
showed at least
some suggestion of a dimeric structure for the
F (
ab’)z p fragment.
Since Green’s review, three new studies were completed which
yielded considerably more detail than had been observed previously.
Shelton and McIntire (1970) and Parkhouse et
al.
(1970) examined the
macroglobulin from the Balb/c mouse myeloma, MOPC 104E, whereas
Feinstein an d Munn (1969) studied yM proteins from several mam malian
species, as well as analogous proteins from chickens and dogfish.
The well-resolved molecule, shown in Fig.
1,
is from Parkhouse
et
al.
(1970) . It is a pentamer made of subunits strikingly like the Y-shaped
structures described for
yG
(Green , 1969 ). T he average dimensions are
- - -
330 350 a *
FIG. 2. Schematic representation of pentameric yM. The dimensions are the
best available estimates from the studies
of
Svehag
et
al. (1969), Shel ton and Mc-
Intire (1970), Feinstein and Munn (1969), and Parkhouse et al. (1970) . Di suEde
bonds a re indicated
by
solid bars. The letters included in the portion of the molecule
illustrating the structure of yM, refer to the peptides described in Table 111. The
filled circles (for clarity shown on only one subunit) indicate
the
areas where
attachment of polysaccharide chains has been implicated.
NH,
indicates the
N -
terminal ends
of
the polypeptide chains. The figure has been drawn with the light
chains ( L ) on the “inside” of the heavy chains ( H ) on the basis of the suggestion
made by Grey (1969a) and in a manner generally similar to that drawn for rabbit
yC by Green (1970).
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STRUCTURE AND FUNCTION
OF yh4
MACROGLOBULINS
69
illustrated in Fig.
2.
Thch 85A. diameter refers to the rim
of
t he F(c ) ~
rings observed by Svehag
et
al.; these rings contained
a
cen tral “hole”
of about
40
A.
The 105
A.
diameter refers to the average distance between
the center of the molecule
and
the ‘‘hinge” point observed
b y
Parkhouse
e t
al.
(1970). The maximum span of the termini is between
115
and
FIG.3.
(T o p ) Schematic moclel of y M with subunits opened to demonstrate the
flexibility about the “hinge” region associated with the disulfide bond which con-
nects the Fab’ regions. (Bot tom) Profile view of model of y M antibody bound to
a particulate antigen. (From Feinstein and Munn,
1969.)
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70 HENRY METZGER
150A. (Shelton a n d McIntire, 1 97 0). These dimensions m ust be con-
sidered provisional. Although the overall diameter is probably reasonably
correct, the internal distances illustrated could b e in error
by
as much as
5M, in part because t he planarity of the structures is so difficult to a ssess.
As discussed by Green (1970) and by Feinstein and Munn (1969),
the structures observed for the free-lying yM are consistent with the
nominally quite different appearing pictures of yM bound to antigens
(H um ph re y and D ourmashkin, 1965; Feinstein a n d M unn, 19f36; Almeida
et
al., 1967; Svehag an d Bloth, 1967; Feinstein an d Mu nn, 196S), if one
assumes that in the latter case the view is from the side. This would
mean that the functional yM contains multiple hinge points resulting in
a structure much more flaccid than that suggested by Figs.
1
and
2.
A
possible configuration is illustrated in Fig. 3.
Occasional hexameric m olecules ha ve been seen b y almost all workers.
That these may not be artifacts is suggested by the finding that the high
molecular weight immunoglobulin of the frog Xenopus
leui
is uniformly
a hexamer (Parkho use
et
al.,
1970 ) ( see Fig.
4).
W heth er su ch molecules
ultimately result from a deviation
of the normal mammalian type of
biosynthesis or from a unique
p
chain (s ee Section
VI1,A)
is not known.
FIG.4.
Electron micrograph of hexaineric immunoglobulin isolated by Dr. R.
M. E. Parkhouse from Xenopus Zed.
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STRUCTURE AND FUNCTION
O F YM
MACROGLOBULINS
71
B. CHEMICALROPERTIES
1 .
Amino Acid Composi t ion and Ni trogen Content
The aniino acid composition of yM immunoglobulins is unremarkable
when compared to other globular proteins and especially when com-
pared to other immunoglobulins
( Metzger
et
a ?.
1968) (see Section
IV,B,4,b).
Nitrogen determinations have been published in only three instances
of which I am aware. For a Waldenstrom macroglobulin containing
10.2% arbohydrate, 14.5% itrogen was measured by Kjeldahl analysis
(F.
Miller and Metzger, 1965a). Human yG and human serum albumin
each gave
16%
itrogen in good agreement with literature values,
A
value
of 14.1%was reported for a Waldenstrom macroglobulin by Jirgensons
(1960). The value of 13.2%eported by Fisher and Canning (1966) for
rat macrogIobulin seems exccssively low.
2. Carbohydrate Content
It is well known (Kunkel, 1960) that y M immunoglobulins have
a
higher carbohydrate content than
yG
immunoglobulins, Innumerable
carbohydrate determinations have been performed on these proteins, bu t
th e value of ma ny of these is limited by t he ina de qu ac y of th e analyses.
Protein concentrations were often determined with inadequate precision
and hexose determinations took no cognizance of the variation in color
coefficients for different hexoses. Even more precise compositional
measurements, however, by theniselves lead to little insight. Only recently
has clarification of the numbers, points of attachment, composition, and
structure
of
th e oligosaccharide chains be en attem pted .
Davic and Osterland (1968) surveyed the carbohydrate content of
eight Waldenstrom macroglobulins. Five of the proteins had an overall
carbohydrate content similar to previously determined values ( 9-13%)
but three had somewhat less ( 7 4 % ) .Concentrations for all eight y M
preparations were based on a single Folin standard, and it might at first
be suspccted that the differences could be due to errors in the deter-
mination of protein concentrations. However, since the decreased carbo-
hy drate content was largely accounted for by a sh arp decrease in mannose
and galactose (with no definite decrease in hexosamine or sialic acid,
and only a small decrease in fucose content), this objection can be
dismissed.
N o
correlation with light-chain class was found.
Glycopeptides from two proteins were obtained from a Pronase digest
by gel filtration and gradient elution from diethylalninoethyl cellulose.
Three distinctive glycopeptides were derived from each of the proteins.
Davie
and
Osterland's da ta are reproduced in Ta ble IIA. Needless to say,
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72
HENRY
METZCER
TABLE
I I A
'ro
DAVIE
N D
OSTERLAND~
COMPOSITION O F hlACROGLOBULIN CARBOHYDRATE UNITS ACCORDING
Glycopeptideb
,yM
I
I1 I11
Group I macroglobidins
Mannose
6-Deoxygalact.ose
Galactose
2-Acetamido-2-deoxyglucose
N-Acetylneuraminic acid
Mol.
w t .
(calculated)
Unit,s/890,000 gm.
IJiiits/heavy chain
Group 11macroglohiiliiis
Mannose
6-Deoxygalactose
Galactose
2-Acetamido-2-deoxyglucose
N-Acet,ylneuramiiiic acid
Mol. w t . (calculated)
Units/89O, 000 gm.
Units/heavy chain
.
0
0
2 . 0
1 . 0
0
1,100
10
1
2 .0
0
1 . 0
2 . 5
0
1,100
10
1
9 . 0
0
1 . 0
2 . 0
0
2,200
1
0
1
2 . 0
1 .0
3 , 5
2 . 5
0
1,700
10
1
6 . 0
2
. 5
2 . 6
5 . 5
2 .0
3,800
20
2
5.0
1 . 0
1 . 0
6 . 5
1 . 0
2,800
20
2
a
From Davie and Osterlaiid (1968).
b Residues per unit to nearest one-half integer.
each
of
the glycopeptides listed could itself represent a mixture. Recent
results by Davie and Osterland
(1969)
suggest that glycopeptide I1 may
be derived from the light chains. Thus, their data indicate at least three
points of carbohydrate attachment to the heavy chains. Studies on the
high molecular weight proteolytic fragments of yM have
also
suggested
at least three points
of
carbohydrate attachment (Section IV,C).
Spragg and Clamp (1%9) examined three type-K Waldenstrom
macroglobulins. They separated their glycopeptides from
a
Pronase
digest by gel filtration, paper chromatography, and high-voltage paper
electrophoresis. Based on the mannose content, two of their three proteins
would be of the Type I1 proteins described by Davie and Osterland.
Except for the high mannose content in the third specimen, it also looks
suspiciously like a Type
11
protein. Although up to eleven glycopeptides
were seen per protein, their data suggest two basic types, roughly similar
to
glycopeptides Types 2 and 3 previously described
by
Dawson and
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STRUCTURE A N D FUNCTION OF yM MACROGLOBULINS
73
Clamp (1968) for a
yA
myeloma pro tein. Th e compo sitions are consistent
with Type 3, being a mixture
of
Davie and Ostcrland's T y p e I a n d 11,
and Type 2 similarly being
a
mixture of Types
I1
and
I11
(T a b l e
I I B ) .
Their d ata suggest four to five carbohy drate chains
per
chain. Molecular
weight data on tryptic glycopeptides from a Waldenstroin macroglobulin
led Bourrillon and Razafimahaleo
(
1967) to a similar conclusion.
TABLE I I B
cOMP OS ITION
OF hIACROGLOBULIN
C A R I 3 O H Y I )R A T E
(;NITS
A C C - O R D I N C :
'1'0 S P R A G G AND
CLAMPn
Glycopeptide
3 2
"fhI
Msnuose 3-6
6-1
)eoxygalactose 0-
1
Galactose
0-
1
2-Acetamido-2-deoxyg111co~e
1-3
A7-Acetylneiirurniii c scid 0
Unih/890,000 g m . 20
3-4
1
1-2
3-4
0-2
30
_ _ _ _ ~ ~
11 From Spragg :tnd Clamp (1969).
Considerably more work will be required to delineate the site of
attachment, composition, and structure of the carbohydrate moieties on
yM proteins. It will be interesting to learn whether the heterogeneity of
carbohydrate content is a reflection of heterogeneity in the amino acid
sequence of the ,p-chain common region; if
so,
this would indicate the
presence of multiple p-chain genetic loci (Section VI1,A).
There is no glycoprotein for which the function of the carbohydrate
moieties has been discovered. The yM proteins are no different. The
extraordinary changes in transmembrane transport seen after minimal
alterations in
the
carbohydrate portion of ceruloplasmin (Morel1 et al.,
1968) suggest interesting experimental approaches.
IV. Subunits, Polypeptide Chains, and Proteolytic Fragments
A.
REDUCTIVEUBUNITS
1.
Method of Isolation
Deutsch an d Morton
(1957)
an d Glenchur
et
al.
(1958)
first described
the formation of stable subunits by reduction of yM with thiols and
reaction with alkylating reagents. Structural analysis of these subunits,
YM,, is complicated by the susceptibility of intrasubunit disulfide bonds
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74
HENRY METZGER
to cleavage under the conditions commonly used to break the inter-
subunit bonds (e.g., 0.1 M 2-mercaptoethanol). Since the constituent
chains are bound to each other by relatively weak noncovalent forces
(Deutsch and Greenwood, 1960), partial dissociation of the subunits
can occur leading to difficulties in molecular weight determinations.
The amount
of
dissociation may vary with the nature
of
the reducing
reagent, alkylating reagent, and unknown factors. For example, Kishimoto
et
al. (1968) remark in a footnote that they failed to see any dissociation
of
,U
and K chains in their study even though their conditions were not
unusual. On the other hand, Suzuki and Deutsch (1967) showed that
by
repetitive gel filtration the subunits could be progressively depleted of
light chains.
Frank and Humphrey (1968) reported a different form of dissocia-
tion, In gradient centrifugation studies with low concentrations
of
iodinated anti-Forssman antibodies, they observed a major peak which
sedimented at a rate consistent with half-molecules of
yM,.
Similar
observations are cited by Harboe
et
al. (1969).
Several investigators have prepared subunits in which the constituent
polypeptide chains remained disulfide linked. F. Miller and Metzger
(1965b) found that with low levels of cysteine (0.01-0.02 M ) at pH
8.6, both inter- and intrasubunit disulfide bonds were cleaved. Re-forma-
tion of intrasubunit p-p and p light-chain disulfide bonds upon prolonged
incubation led to complete conversion of the y M polymer into subunits
the polypeptide chains
of
which were not dissociable. Mihaesco and
Frangione ( 1969), using slightly different conditions of reduction with
cysteine, obtained selective cleavage of the intersubunit bonds. Mer-
captoethylamine was successfully used by Morris and Inman ( 1968).
They showed that approximately two -SH were released per mole
of
subunit formed (based on a subunit molecular weight of 180,000). No
chain dissociation occurred under denaturing conditions. Beale and Fein-
stein (1969) have secured noildissociable subunits by reduction of yM
at very low ( 0.000125 M ) dithiothreitol concentrations, but qualitative
data suggested that one of the intrasubunit disulfide bonds was cleaved
in at least some of the subunits.
Subunits are most often isolated on Sephadex G-200 or Bio-Gel P-200.
On diethylaminoethyl cellulose they are eluted at lower ionic strengths
and higher pH’s than the parent y M (Reisner and Franklin, 1961).
2.
Physical
Properties
Even where substantial chain dissociation was avoided, differences
in the molecular parameters have been reported. F. Miller and Metzger
(1965a) measured a molecuIar weight of 185,000 and calculated a
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STRUCTURE AND FUNCTION OF y M MACROGLOBULINS
75
molecular we igh t of 178,000 from
a
pentameric model for yM of molecu-
lar weight 890,000. Inman an d Hazen (19 68) reported a value of 177,000
for their subunits, and Feinstein and Buttress (1970) have obtained
181,000. Lamm an d Small (196 6) obtained a molecular weight of 180,000
for th e subunits of rab bit yM, although som e uncertainties were e n-
countered in those studies. Suzuki an d D eutsch (1 96 7) , on th e other
hand, estimated the molecular weight of their subunits
as
200,000. Since
the latter workers obtained a molecular w eight of 1,000,000 fo r th e
unreduced yM, they also concluded that the yM was a pentamer. Filitti-
W urmser an d Ha rtm ann 1968b) obtained subunit molecular weights
that ranged from 1.4 to 1.9
X
1oj daltons. Since the parent yM molecular
weights varied independently of the subunits, thcy proposed that yM
molecules may contain 4, 6, or 8 subunits alternatively.
The subunits produced by reduction fully account for the chemical
composition of the whole yM and, ordinarily, reduction leads to no loss
of peptide
or
carbohydrate material (se e Section IV ,C,3 ). T he subunits
have a slightly greater anodic mobility than the parent molecule
(McD ougal l and Deutsch, 1964 ) , and
a
small change in the spectro-
photometric titration curve has been reported ( McDougall and Deutsch,
196 4). T he latter might have been du e to partial chain dissociation. Th e
partial specific volume a nd extinction coefficient at n eu tral p H ar e un-
changed (F. Miller a nd Metzger, 1 96 5a ). Th ere is a marked drop in the
intrinsic viscosity of t he yM prep aration upo n red uction [0.162 to 0.080 in
the study of F. Miller and Metzger (1965a)l and, in general, the sub-
units behave as more compact units th an do t he p aren t molecules.
Occasional antisera distinguish between subun its and p are nt m olecules
(Korngold and Van Leeuwen, 1959; Reisner and Franklin,
1961;
W O K
heim and Williams, 1966; Swedlund et al., 1968; Gleich and Loegering,
19 69 ), b u t sometimes this m ay simply reflect the antigenic polyvalency
of the pentamer compared to the monomer, rather than unique deter-
minants on the former (Gle ich an d Loegering, 196 9).
3. Specific Reassociation
Reduced yM preparations contain subunits and polypeptide chains
with free sulfhydryls, so it is not surprising that reaggregation should
occur on reoxidation. Similarly, since in many assays activity is enhanced
by polyvalency (Section VI,A,3), it is not surprising that m any investiga-
tors were simultancously able to recover binding activity. Of greater
interest is the highly specific reaggregation
of
the subunits which can
be
obtained under app rop riate conditions (Park hou se e t a l . , 1970; Inm an,
1969) . That is, the subunits reassociate into pentameric y M polymers
with only very low levels
of
intermcdiatc-sized or heavier than 19 S
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76
H E N R Y
METZGER
aggregates being formed. This observation complements the finding that
even un der conditions wh ere only some
of
the polymer is reduced, little
or no intermediate products are formed. Both results demonstrate the
preferential stability of th e pentanieric po lymer. It was this phenomenon
that first suggested to F. Miller and myself (1965b) that the polymer
was a closed (i.e. , circ ula r) rather th an a linear structure.
B POLYPEPTIDE
HAINS
1.
Methods
of Isolation
T h e yM proteins dissociate into heavy and light chains in much the
same way
as
yG immunoglobulins (Cohen,
1963;
Carbonara and Here-
mans, 1963). Under reducing conditions adequate for breaking all the
interchain disulfide bonds (see F. Miller and Metzger, 1965b), the re-
leased sulfhydryls may be blocked with a variety of reagents and the
chains separated by gel filtration. Sephadex G-100 or Bio-Gel P-150
equilibrated with 1
N
acetic or propionic acid are most often used. In
my own experience (using 1N propionic acid and Sephadex G-loo),
maximal resolution is achieved a t pro tein loa ds
no
greater than about
1
mg./lO ml. be d volume. Heavy chains sep arated in
1M
propionic acid
do not release any further light chains on subsequent rechromatography
in
7 M
guanidine (Metzger, 1969b)
and by several criteria are not
substantially contaminated with light chains (Putnam et al., 1W 7) . For
separating chains in which intra- as well as interchain disulfides have
been cleaved, good results ar e obtained with Se phadex G-200 equilibrated
with 5 to 7 M guanidine (L am m a nd Small, 196 6).
2.
Yields of
Chains
Accurate determination
of
the yields
of
light and heavy chains
depends on the adequacy of chain separation. Thus, many values listed
in the literature can only be considered approximate since the elution
patterns show inadeq uate separation or are no t described. I n our separa-
tions we usually find 22% of th e total recovered 280 mp. absorbancy units
under the light-chain peak and have never observed a yield higher than
27% T he extinction coefficients of t h e light a n d heavy chains in prop ionic
acid are, in our hands, approximately equivalent ~ Z o e ; ~ ~ . .2) S O that
the absorbancy yields approximate the mass yields. Considering the
molecular weights of the chains (below) the data are most consistent
with a yield of one light chain for each heavy chain, The results of
disulfide cleavage (below) are in support of this. Suzuki and Deutsch
(1967) reported a considerably higher yield of light chains. Their
separations were performed
on
Sephadex G-100 and in
5
M guanidine.
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STRUCTURE
AND FUNCTION OF
yM MACROGLOBULINS
77
A 32% yield of a bsorbancy was re cord ed for th re e different pr0teins.l
Lamm an d Small (1 96 6) stud yin g rab bit yM, obtained 22-23% of the
total absorbancy under the light-chain peak.
3. Properties of
Light
Chains
Early studies
on
the amino acid composition, molecular weight, and
light-chain genetic markers consistently showed that as a group, the
light chains associated with yM immunoglobulins were not distinguish-
able from the light chains associated with other immunoglobulins. An
exception to this statement are the data on
~ : h
ight-chain ratios for yM
relative to other immunoglobulins. Wollheim and Snigurowicz ( 1967)
observed only twenty-five h proteins am ong 12 5 macroglobulin “M” com-
ponents, whereas among polyclonal normal macroglobulins the h
:
ratio
was more like 1:2. In an extensive series studied from November, 1966
to March, 1969 th e National Cancer Institute Im mu noglobu lin Reference
Cen ter recorded only twenty-two proteins in
a
to tal of 139Waldenstrom
macroglobulins for which the light-chain typing was unambiguous
(Wood s, 19 69) . Th e percentage of
h
proteins ( 17 % ) is only ab ou t half
that observed with yG (36%)and yA
( 31%)
mmunoglobulins. Recently,
N-terminal amino acids (C oh en an d C ooper, 1968; Putnam e t
al.,
1967)
and twelve partial N-terminal sequences ( N iall an d E dm an, 1967;
Edm an and Cooper , 1968; A. P. Kaplan and Metzger, 1969) have been
determined on light chains from yM proteins. No distinctive features
were observed in these studies. Solomon and McLaughlin (1969) have
described an antiserum that distinguishes between
K
subgroups-i.e.,
K I and KII subgroups which end in aspartic acid and K ~ ~ Ihich ends in
glutamic acid (Hood an d Talniage, 1 97 0). Nine o ut of ten K chains from
yM w ere said to b e of th e K~~~ subgroup by Ouchterlony analysis, whereas
on l y 2040%of non-yM light chains were of this type. Neither the N-ter-
minal data of Putnam et al. (1967) nor of A. P. Kaplan and Metzger
(1969)
supp ort such a skewed distribution: th e K~~~ subgroup w as present
in only two of the ten K chains reported on in those papers. That three
of four K light chains from yM cold agglutinins were of the K ~ I I ubgroup
(Cohen
an d C ooper, 1% 8) may simply represent a sampling “error”2 or
Dr.
H.
F. Deutsch kindly sent me the protein VI
(Suzuki
and Deutsch, 1967)
to examine in my own laboratory. My stndies indicate that our different findings are
more related to differences
in
the experimental techniques than
to
any differences
in the proteins used.
Thu,;,
I obtained only a 27% yield
of
light chains, and enuniera-
tion of the
clisulfide
bonds gave results in agreement with our previous results
(F.
Miller
a n d
Metzger,
19651)).
’
f the incidence of K ~ , , s 30%, he prol~abilityof obtaining three K l l , sulyqroup
chains in a random set of
four
is
7 .6%.
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78 HENRY METZGER
a reflection of the activity being selected for (Section VI1,A). Clearly,
furth er studies are re quire d to clarify the app aren t discrepancies.
4 . Properties of Heavy Chains
On the basis of molecular weight data for the yM polymer
and yM, monomer, an equivalent number of heavy and light chains, and
gel filtration data, F. Miller and Metzger (1965) suggested that the
p
chains had a molecular weight of between 65,000 to 70,000. Experimental
support was simultaneously obtained by Lamm and Small (1966) who
studie d rabb it chains and obtained a molecular weight of
+70,000.
T he majority of detailed direct determinations performed since the n
(Suzuki and Deutsch, 1967; Dorrington and Mihaesco, 1970) have
supported these figures. The molecular weight of the chains can also be
determined by amino acid composition in the following way: The
cysteines contributing to the interchain bonds can be selectively cleaved
(s ee below ) and th e sulfhydryls alkylated with '*C-iodoacetamide of
accurately know n specific activity. Since the nu m be r of carboxym ethyl
cysteine groups per chain is known (below) a known number of moles
of heavy chain can be applied to the amino acid analyzer. The summed
amino acid composition should yield the molecular weight. In a recent
experiment (Metzger, 1969b) the molecular weight (for the carbo-
hydrate-free portion of the p chain) was calculated to be +60,000. With
the carbohydrate added this would bring the total weight to between
68,000 and
70,000.
A similar determination on th e light chain g ave
22,400.
Molecular weights ranging from 49,000 to 72,000 have been deter-
mined on p chains from three different Waldenstrom macroglobulins
( Filitti-Wurmser et al., 19 69 ). O ne would like to see additional experi-
mental approaches applied to these materials in order to verify these
results. The rather high molecular weight
(
75,000) obtained by Bennett
(1969) rests on somewhat shaky experimental grounds, although
a
sub-
stantially different experimental approach by Habeeb et al. (1969 ) led to
the same result. Since considerable progress is being made on the full
sequence of p chains (Wikler et al., 19 69 ), formula weights should soon
be available so that belaboring this point seems unproductive.
b.
Amino Acid Composition.
The amino acid composition of
p
chains is unremarkable when compared to the composition
of
other
immunoglobulin chains, Indeed, when examined statistically (Metzger
e t
al., 1968) heavy and light chains of several classes are remarkably
similar with respect to their amino acid composition per unit weight.
W hen calculated in this way (w hic h obviates all assum ption s), i t is
clear from several detailed amino acid compositions ( Suzuki and Deutsch,
1967; Putnam
et al.,
1967) that such d ata
cannot
be used to build molecu-
lar models-i.e., any number of l ight and/or heavy chains
of
any
a.
Size.
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STRUCTURE AND
FUNCTION
OF
-yM MACROGLOBULIR'S
79
assumed size would give substantially the same composition for the
y M .
c.
Sequence
Data.
The following data are available: the C-ter-
m i d nine amino acids
of
several
p
chains (VC'ikler
et
al.,
1969;
Grey,
1969'17); a sequence of sixteen amino acids apparently homologous with
TABLE 111
P R I M A R Y 8TRITCTITRE OF p CIIAINS: THE I N T E RCH A I N
DISULPIDERII)QESn
~~ ~
-
1" C H O 7230
1
I 1
(NH,-j . .
. (.
.
. P-L-V-S-CJ-Z-I3-S
. .)
(G-L-T-F-Q-Q (N)A-S . . CH . . It j
.
.
105 1 0 21
A= 13
C
-
t
b
Y M O
* 1
. .
(.
. CrI,I,E
. .)
. . . (. . h[-S-]~-'1'-A-C-'l'-CIT-Y-C[)OII)
22 21
D
E
b
The one-letter
amino acid code
is used (Thyhoff, 1 %9):
A = Ala
I
=
Ile S
=
Ser
B
=
Ass
K
=
I,ys
T
=
Thr
c = c y s
1, = Leu
V
= Val
D =
Asp
RI = 31et.
w
=
'rrp
E = Glll N =
As11
x =
llrlkllowll
F = Phe P =
Pro
Y = Tyr
H = His
G
=
G l y
Q
=
Glll
z =
GI11
01'
G l X
Ii
= Arg
*
Numbers above t>hecysteitie residiies refer t.o approximat>eposition of homologoiis
regions
iii
y
chains (Edelman
el ul.,
1969). The total length
of --530 residiies
s
based on a
presumed polypeptide weight of
58,000
gm.
and
a
mean
residue weight of 109 gm.
Peptide isolated from
p
chairi:
(A)
N-Termiiial sequence of
105
amino acids deter-
mined
oti
protein
Ou
hy
Wikler
et
a,l.
(1969). (B)
Chymotryptic peptide sixteen residues
long which contains the cysteine
(CL)
which forms the p light,-chain
bond.
Sequence
shown is
by Pink arid hlilstein
(1967). Cnmpositiori
of
this peptide (Beale
and
Biit,tress,
1969) shows, in addition, B1,
TI,
Sr, ZI, G I ,
A I ,
V,. ( C ) Tryptic glycopeptide t,wenty-orie
residues long released during
Fab' p -+ Fall p
coiiversioti
(Section IV,C,2,b) (Met,zger
d
ul.,
1966h; Beale atid But tress, 1969). The shor t N-1.ermirialseqiiencewas obtained
by
Edmaii
degradation (Metzger
et tzl.,
1966h). Th e
asparagiiiyl-carbohydrate in
position
7
i s by a
variet,y
of
inferential
biit,
not tlirect,
evidetice.
This peptide
cont,ains,
iii addit,ioii
to the cysteirie (Crr)contribiitiiig ( o (he p-p bond in E'(ab')n, C-termiiial arginine
atid
Un, T I , SI,
Zl,
PI,
A,,
VI, R I , , I,. ( I ) ) 'I'ryptic.
glycopept,ide ohtailled from papaiti
Fc p
fragment,. It, has a variahle s rnoun t of carbohydrat,e
atid
the cysteine (*) forming the
p-p
iiitersubririil hridge (Beale aiid Biittress, 1969). I t s positioii
is
utic.ert:titi (see text);
Its
composition is C,,
B3,Tr, 83, Z3,PI,
G2A2V111FIEIlRl.
F)
C-Terminal tryptic
glyco-
peptide (Heale arid Buttress, 1 169) which cotitnitis the C-terminal
ryaiiogeii
bromide
Eragmeiit
seqiieiiced
hy Wikler ct ul.
(1 )69)
and
by
Grey (19691,).
The
C:-lermirial
se-
quence A4- ' l ' -C-Y w : ~lso obLaiiied from p chaiiis by Ahel and Grey (1 )67).
Ii i
itddi-
tiori to the amino :wids shown the peptide cotitailis B I , TI,
i,,
Z,, I'l, GI,
V?,
Ln,
HI.
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80
HENRY METZGER
an Fc peptide of rabbit y chains (Wikler et al., 1969);
a
composition
and partial sequence of a tryptic peptide from the hinge region (Beale
and Buttress, 1969; Metzger
et
al.,
196613) (T a b le 111); th e se que nce of
a
short region which includes the p-chain cysteine participating in the
p l ight-chain disulfide bond (T ab le IV ) ; a stretch of 105 amino acids
from th e N-terminal e nd of o ne yM (Wikler et
al.,
1969 ); sh ort sequences
from the N-terminal end of nine other
p
chains (Bennett, 1968; Kohler
et al., 1969; A. P. Kaplan et al., 1970) (Table V ) ; N-terminal amino acids
from many p chains; and tryptic fingerprint data from many proteins
(Putnan i et al., 1967; Franklin and Frangione, 1968; Bennett, 1968).
These d ata pe rm it one to arrive a t the following tentative conclusions:
( I )
A
substantial portion of the p-chain sequence is common to all
p
chains. Though considerably more information must be obtained, the
data that we have are consistent with p chains having an N-terminal
variable region similar in length to that of
y
chains (115-120 residues).
(2) Although th e da ta of B ennett ( 1968) (Table
V )
suggest tha t p-chain-
specific variable regions may exist, the data of Wikler et al. (1969) and
A.
P. Kaplan e t al. (197 0) app ear to indicate qu ite the opposite. Tha t is,
there appears to
be
remarkable homology between the variable region of
p a nd
y
chains (t h e variable region
of
all subclasses of human y chains
are
so
far indistinguishable). Indeed, the p-chain sequence reported by
TABLE IV
HEAVY-LIGHT-CHAINYSTEINE",~
P R I M A R Y SI'RLICTURE OF p
CHAINS:
IIOMOLOGIES A R O U N D
~
125 130 135
P P-L-V-S-CL-Z-B-S Pink arid Milstein
(1967)
Yl
(1969)
Y2 P-IA-A-P-CL-S-R Frangione
et
al.
(1969)
Y3
P-L- A-P-CL-S-It Frangione and
Mil-
stein (1968)
Y4 G-P-S-V-I-P-L-A-P-CL-S-R-S-T-S-E(S,T)-A-L Pink and Milstein
(1967)
Y
A-P-8-V-F-P-L-A-P-CL-C-G
OWonnell el al.
(Rabbit) (1970)
G-P-S-V-F-P-I-A-P-8 - 8-K-8-T-S-G-G-1'-A-A-L Edelman cf al.
Y l V-E-P-I-S-CL-I
)-K-T-C Edelman et al.
(1969)
220
As iii Table 111,
the
one-letter amino acid code is used. The niinbering is based
on
(CL)
Cysteiiie implicated
i t i
hettvy-light,-chaiti disrilfide bond.
the coinp1et.e seqiieiire
of
t.he
7 1
chuin
of protein
Eli (Edelman c1
al.,
1969).
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STRUCTURE
AND FUNCTION
OF
yM MACROGLOBULINS
81
TABLE V
PRIMARYTRUCTUREF p CHAINS:COMPARATIVEEATURES
OF
N-TERMINALEGIONS
Heavy- Position
Sub- Chain
group” Protein Class Reference 1
2
3
4 5
6 7
8
9 1 0
V H I Eu
7
Misc.
proteins
Mar
Wag
How
Lay
Koh
Di
VRII Daw
Cor
He
o u
(Ioc)
VHIll Dos
Bal
Bus
Daw
YI
Y1-1
Edelman et al. (1969) Z-V-Q-L-V-Q-S-G-A-E
Press and Hogg (1969)
A.
P.
Kaplan et al. (1970)
~ H-
-
~
&--
E
A
~
A.
P.
Kaplan
et al.
(1970)
A .
P. Kaplan el
al.
(1970)
A. P.
Kaplan et
al.
(1970)
A. P.
Kaplaii
et d. 1970)
A. P.
Kaplan ct al. (1970)
Kohler et al. (1969)
Press and Hogg (1969) Z-V-T-I-It-E-S-G-P-A
Press and Hogg (1969) ~
Cunningham
d
al . (1970) K-N-T
Wikler d al. (1969)
T
[A.
P.
Kaplan et al.
(19701
Bennett (1968) Z-S-V-A-B
Bennett (1968)
E
L-ennett (1968)
Bennett (1968) L-
-- .
a The data are grouped according t o the guidelines developed a t a WHO nomencla-
ture conference held in Prague, June 1968. Subgroups HI and HI1 are based on the
extensive sequence data for protein
ELI
Edelman
et
al., 1969) and for proteins Daw and
Cor (Press and Hogg, 1969). Protein 011 or which the N-terminal sequence of 10.5 amino
acids
has
been reported shows 70% hornology with the first ninety-nine residues
of
protein Daw but
only 2975
hornology with a comparable stretch from protein Eu.
Subgroup HI11 is based entirely 011 the perltapeptides reported by Bennett (1968).
Wikler
e t al.
(1969) is actually more closely related to the yDrbWequence
(Press and Hogg, 1969) than is the latter to the yEU sequence (Edelman
et
al., 1969). It can be tentatively concluded that p chains may well have
their variable regions coded for by the same group of genes that code
for the variable regions of other heavy chains. Discussion of these data
as well as their implications can
be
found in several recent publications
(Wikler
e t
al. , 1969; Ed elm an and Gall, 1969;
A. P.
Kaplan
et al.,
1970;
Metzger, 1970).
5.
Disulfide Linkage of
y h l Chains
a . Methods of Analysis. Without citing “chapter and verse,”
a
review of recent data from many laboratories convinces me that
( a )
amino acid analyses of carboxymethyl cysteine, cysteic acid, or amino
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82
HENRY
METZCER
ethyl cysteine freq ue ntly give results w hich ar e off by 10% n d often much
more; ( b ) nalyses of SH groups with 5’,5-dithiobis-( 2-nitrobenzoic a ci d )
(Ellman , 1959) can
be
highly accurate but are cumbersome when large
numbers of samples mu st be assayed (e.g., from an elution pa tte rn );
( c )
with care, reproducible an d accu rate results can be obtained w ith
radioactive alkylating reagents. The latter method also permits rapid
assay of large numbers of samples. The additional possibility of using
doub le label ing (w ith
I4C
and ‘H reagents) and radioautographic tech-
niques allows for great flexibility. In my own laboratory we use this
method exclusively when maximal accuracy is required.
Number
of Interchain Disulfide
Bonds. A
detailed study of the
interchain disulfides of
a
human yM protein has been presented
(F.
Miller and M etzger, 19 65 b) , an d only the conclusions will be presented
here since a complete analysis is given in that paper. It was shown that
there ar e 2 4 2 5 interchain disulfide bonds per molecule (890,000 dalto ns)
of
y M .
Four-fifths of the released sulfhydryls appeared under the heavy-
chain peak when the chains werc separated. These data showed that the
light chains were co nnected to th e heavy chains by a single disulfide link,
that there were three inter-heavy-chain bonds, and that one or two
of
the latter formed the intersubunit l inks (cf. Chaplin et al., 1965) . That
the heavy chains contain four cysteines participating in interchain links
has been show n by others. By using preparations in which only interchain
bonds had been cleaved, it was shown that four unique peptides contain-
ing the participating cysteines could b e isolated (Mihaesco a n d M ihaesco,
1968; Beale and Buttress, 1969; Beale and Feinstein, 1969, 1970). The
4
1 ratio of heavy-chain : ight-chain sulfhydryls obtained after selective
cleavage of interchain disulfides shows that there must be
a 1: l
ratio
of heavy :light chains. These d at a a re inconsistent with th e tw o heavy-
chain: three light-chain model proposed
by
Suzuki and Deutsch
(
1967) .
The interchain disulfide bonds can now
be
provisionally placed (Table
111).
The most N-terminal must be that
connecting the light and heavy chains since it occurs in the Fab region
(Section IV,C,2,a)
(F.
Miller and Metzger, 1966; Beale and Feinstein,
1969; Beale and Buttress, 1969). A short sequence around the p-chain
cysteine contributing to this bo nd has been reported by Pink a nd M ilstein
(19 67 ) . There is a strong hint of homology to the sequence around
a
comparable cysteine y..,
y 3 ,
a n d y 4 human heavy chains and in rabbit y
chains (Table
I V ) .
The sequence is quite unlike that around cysteine
220 which performs a similar function on yt chains. Further sequences
will obviously be required to substantiate this homology.
The disulfide bond C-terminal to the former one connects the Fab‘
regions (F. Miller and Metzger, 1966; Metzger et d. 966b; Beale and
b .
c. Locution
of Bonds.
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STRUCTURE AND FUNCTION OF yM MACROGLOBULINS
83
Feinstein, 1969; Beale an d B uttress, 1 96 9) . Th e third relevant cysteine
is
found in peptide p3-p5 of Beale and Feinstein (1969) and Beale and
Buttress
(
19 69 ). Th e fourth cysteine contributing to t he inter-heavy-
chain bonds is
a
cysteine that is the penultimate amino acid residue of
the chain (Ta ble
111).
The latter residue, though contributing to an
interchain bond, is not the one contributing to the intersubunit bo nd. T he
amino acid composition of the peptide thought to contribute to the inter-
sub un it bond by Beale and Feinstein (1 96 9) and Beale and B uttress
(1969) does not contain the C-terminal tyrosine (Doolitt le et al., 1966,
Abel and Grey, 1967; Mihaesco and Mihaesco, 1968) nor the methionine
known to be nine residues from the C-terminal end (Wikler et al., 1969;
Grey, 19 69b ). Furthermore, when only th e intersubunit bonds a re
cleaved a n d 14C -alkylated, the C-term inal pep tide contains no rad io-
activi ty (Grey, 1969 b) and a t r ipeptide (I lc ,Glu,Cys) is obtained
(Mihaesco and Frangione, 1969) the composition of which is incom-
patible with the C-terminal sequence but is clearly compatible with the
composition of peptide
p4-pLn
mplicated by Beale and co-workers. The
recently reported study by Beale and Feinstein (1970) is confirmatory.
The presence of only one intersubunit bond agrees with the conclu-
sions of Morris an d Inm an
(
1968) (Section IV,A,l ). In view of the com-
position of the C-terminal twenty-one residue tryptic peptide
(
peptide E
in Table
111)
the intersubunit bond m ust be a t least tha t far distant from
the C-terminus of the
p
chain. That the intersubunit bonds are relatively
close to th e C-termina l ends of the chains was suggested by Inman
an d Haz en (19 68 ) who observed release of yM ,-sized fragme nts after
brief incubation of yM with papain. These experiments did not, how-
ever, exclude the possibility that these fragments resulted from ( a )
papain scooping out
a
region of p chain containing the intersubunit bond
but lying within an intrachain disulfide
loop
(thereby yieldillg
a
yM,-
sized unit regardless of the points of cleavage) or ( b ) papain acting
as a catalytic reducing agent and not as a protease.
6. Chain Reconibination
Relatively little has been published on recombining the dissociated
chains of macroglobulins. Gordon and Cohen (1966) recorded some
observations on two yM proteins
as
pdrt of a study on chain recombina-
tion by heavy chain$ from various classes. Th ey o bserved preferential
reassociation of autologous chains, similar to the phenomenon exten-
sively docum ented by G rey and Mannik (1965 ) and Mannik
(1967a)
for y chains. E. J. Miller and Terry (1968) described a unique antigenic
determinant on p-K protenis, which could only be recovered by recom-
bining p chains (fro m either x or K proteins) with K chains.
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84
HENRY METZGER
T A B L E
V I
PR O T E O L Y T I C
F R A G M E N T S
OF
yM PROTEIN6
Antigenic
determinants
Hexose SH groups Light-
gm./100 per mole chain p-Chain
Mol. wt,.” gm. after mild deter- determi-
Fragrnen hm b
x
10-4 protein) reduction minant8s nan ts
I.
F a b p
Trypsin
(1-3) 4.1-4.8
3.6-3.7 0.8-1.9
2
All Some
Chyrnotrypsiri C
(4 ) 4 . 0 3 . 8
N.Rd N.R. All Some
Pepsin
( 5 8 )
4.8-5.5 3.3-3.5
1.8-2.8
N.R. All Some
Papain 4, .5 8, 9, 11)
3.7-5.5 3.5-3.8
1 . 6 - 4
2 All Some
Tiypsin
(1-3)
4 . S 6 . 9 4 . 3 - 4 . 4
1.4-2.8
3
All >Fab
Chym ot,rypsiri C (4) -6.8 N.R. 2 . 2
N.R. All
>Fab
Pepsin
(5-7) 6 .3 -7 .3 4 . M . 4 5 .1 -5 .4
N.R. All > F ab
Papain
(5)
7 . 1
6 . 0
3 . 9
N . R . All > F a b
Trypsi11
(1-3)
9.5-14
6 . 0 - 6 . 3 1 . 4 - 2 . 8
6
All =Fab’
Chyrnotrypsin C
(4)
14 6.6
2 . 2
N.R. All =F ab ’
Pepsin
(6-7)
12-13 5.6-6.6
5.1-5.4
N.R.
All
= F a b ’
Papain (4, 5, 9, 10 ) 13 7 . 0
3 . 9
N.R. All =F ab’
Trypsin (3)
6 . 7 3 . 4
12 N.R. None
Most
Papain
(2, 8-12)
3.2’ 2.9-3.2
22 2/32,000
None
Most
Trypsin
( 3 ) ~ 34 11
12
N.R. None
Most
Papain
(8, 10)
32
11
22 18
None
Most
11.
Fab’
p
111.
F(ab’),
p
IV. Fcpe
V.
F(c)bp
a Numbers in parentheses indicate the following references:
(1) F. Miller an d M etzger (1966)
(2)
Beale and B uttress
(1969)
(3)
Plaut and
Toxasi (1970)
(4) Chen el
a l .
(1969)
(5)
Sueuki
(1969)
(6) Mihaesco and Seligman (1968a)
For nomeiiclalure bee text.
All molecular weights an d sedim entation coefficients hav e been round ed
off
to
two
(7) Kishimoto et al. (1968)
(8)
Dorrington and Mihaesco
(1970)
(9)
Onoue
el al. (1967)
(10)
Onoue
el al. (196813)
(11)
Mihaesco arid Seligman
(1968b)
(12) Mihaesco and Mihaesco (1968)
significant figures.
2 N . R .
=
not reported.
Obtained by reduction of F(c)s
p .
f
Analyzed in
5
M
guanidineHC1.
Tr yp tic digestion performed
at
high temp erature. Similar results said to be obtain-
able using bovine
01-
or @-chymotrypsin(Plaut a nd Tomasi, 1970).
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STRUCTURE AND FUNCTION OF
YM
AMACROGLOBULINS
85
It would b e of som e interest to s tud y the recombination of chains from
Waldenstrom macroglobulins with defined binding activity.
C.
Early reports by Petermann and Pappenheimer ( 1941), Deutsch
et a2. (196l) ,and H arboe (1965) suggested that macroglobulins might
be
fragmented by pepsin and papain in much the same way that r G im-
munoglobulins can b e cleaved. Only more rccently, how ever, have precise
chemical data been collected on such fragments. Table V I gives some of
the results from studies with a variety of enzym es. Th e tabIe is not me an t
to be exhaustive.
PROTEOLYTICRAGMENTSF yM MACROGLOBULIN
1 . Nomenclature
When we performed our s tudies
on
the tryptic digestion of TM
proteins (F. Miller and Metzger,
1966)
the question of nomenclature
arose. The 50,000-1nol. wt. fragmen ts p rod uc ed from yG by papain in
the presence of mild reducing agents had been called Fab fragments,
whereas the dimeric
110,000-mol.
wt. fragments produced by pepsin
cleavage were called F(ab') , fragments . We decided to focus
on
the
structure of such fragments rather than
on
the proteolytic enzyme
employed an d for this reason ad opte d the following rule: Any fragment
consisting of a single heavy-chain fragment and a more-or-less complete
light chain, an d having o ther properties analogous to the p apain fragment
of yG, would be called Fab regardless of the enzyme used. The terms
F(
ah')? (a n d its reduction produ ct, Fab ') were similarly used even wh en
a
protease other than pepsin was employed. In conformity with the
guidelines developed at a nomenclature meeting ( World H ealth O rgani-
zation, 1964) a
suffix
denoting the heavy-chain class of the fragment was
added, e .g. , Fab p. I continue to feel that this is a reasonable approach
and have organized the data in Table VI accordingly.
2. Types of Proteolytic Fragments
I t
is
clcar from Table
V I
that fragments closely analogous to those
obtainable from
7G
proteins can be obtained from yM immunoglobulins.
Th oug h not indicatcd in the table, the assignments ma de on the basis of
structural criteria were completely substantiated when functional macro-
globulins were employed; that is, the Fab fragments contained the
combining sites ( Section
VI,A,2).
Fab Fragment. A relatively stable fragm ent is obtained by pro -
teolysis with trypsin, chymotrypsin
C,
an d papain under the usual condi-
tions. The susceptibility to more rapid breakdown during hydrolysis with
a.
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88 HENRY METZGER
pepsin (Mihaesco and Seligman, 1968a; Kishimoto et al., 1968) is
probab ly related to th e relatively w eak heavy-light-chain interactions
at the low
pH’s
required for peptic activity. This is supported by the
increased susceptibility of these fragments to papain at similarly low
pH’s
(
Mihaesco and Seligman, 1968b).
It was possible to estimate th e yield of F a b fragments in several
studies. From the extinction coefficients, molecular weights, and ab-
sorbancy yields, F. Miller and Metzger (1966) showed that precisely
2 moles of Fab were produced per mole
yM,
(based on a molecular
weight of 180,000 for th e lat ter ). Similar yields w ere ob tained by Ono ue
et al. (1967 ) using papain a nd by Chen et al. (1969) using chymotrypsin
C. This is evidence against
a
hypothetical model recently proposed by
Suzuki (19 69) . Th e latter m odel was intended to show how the pro-
teolytic fragmentation data might
be
consistent with the five-chain model
of Suzuki an d Deutsch (1 96 7). I n fact, all th e fragmentation data along
with the disulfide cleavage studies
(
Section IV,B,S,b
)
provide strong
support for a four-chain, symmetrical subunit.
It is unlikely that the Fab p fragments really have as wide a range
of
molecular weights
as
is indicated in Table VI. Sequence data will ulti-
mately provide definitive values.
T h e
Fd p
fragment ( t h at part of the heavy chain in the F ab fra gm ent)
can be separated from the light chain by repetitive gel filtration
(F.
Miller and Metzger, 1966; Onoue et al., 1967). It may be a useful inter-
mediate for future sequence studies though the Fd’ @ fragment (see
below) can
be
purified more easily.
Fab’
p
Fragment. This, by definition, requires both reduction of
the
p-p
bond present in the F(a b /) )? region as well as scission of th e
p-chain C-terminal to tha t bond. It is
a
useful fragmen t. I t can be isolated
in up to 85% ields from a 15-20 minute tryptic digest of yM,. T he Fd’ p
piece can then be isolated by gel filtration of th e Fab’ on Sep had ex G-100
in 1
M
propionic acid
(F.
Miller and Metzger, 1966; Metzger e t al.,
19 66 b). T h e separation of the
Fd’
(w hic h should be a useful intermediate
for sequence studies) from the light chain is much superior to the
separation achieved with Fd piece and light chain.
If Fab‘ is redigested with trypsin, a single, nonultraviolet-absorbing,
glycopep tide is released of m olecular w eigh t 27OOO-8000, containing
twenty-one amino acids and the cysteine contributing to the
p-p
disulfide
br idge in
F(
ab’), (Metzger
et
al.,
1966b). Its composition is identical
to that for peptide ,u2 reported b y Beale and Buttress (1969 ) (se e Table
111).T h e prese nce of this hinge region is essential for
an
important anti-
genic determinant on the p chain-an observation confirmed by all who
have investigated antigenic differenccs between
F(
ab’) ? and Fab f rag-
b.
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STRUCTURE AND
FUNCTION
OF
yM
MACROGLOBULINS
87
ments. It
is
of interest that this hinge peptide has only a single proline,
whereas cysteines participating in inter-heavy-chain bonds in various
y
chains are commonly surrounded by multiple prolyl residues
(
Frangione
e t
al., 19 69). It is tantalizing to speculate that this may make the hinge
region of yM proteins less flexible and that this, in turn, might make
expression of divalency more difficult ( Section VI,A,Z),
F (
ab ), Fragment.
As with Fab fragments a tremendous varia-
tion in molecular weights has been reported (Table
I ) .
Whether this is
due to variability in the point of p-chain scission, carbohydrate content,
or experimental error, is uncertain.
F c p and
F ( C ) ~ ~
ragments.
Th e F c p fragments were f irst iso-
lated by Mihaesco and Seligmann
(
19 66 ), Seligmann an d Mihaesco
(1967 ) , and Onoue
e t
aZ. (1967) from papain digests of yM. Mihaesco
and Mihaesco (1968) confirmed the presence of the C-terminal tryosine
an d cysteine in such preparations. Dorrington an d Mihaesco (197 0) con-
firmed the molecular weight of 320,000 originally reported for
F(
c),
p
by Onoue e t 01. (1968b) and showed that after reduction and dissocia-
tion in guanidine-HC1, fragments of 32,000 were released. Similarly, a
340,000-ni01. wt. fragment was obtained by P lau t an d Tom asi (1 97 0)
from high-temperature tryptic digests. After reduction and study in a
nondissociating solvent, homogeneous fragments of 67,000 mol. wt. were
found. These results confirm the presence of ten chains pe r mole y M .
Adding the weights of the
F(
ab')s p fragments to those of the
F(
c ) , p
the data of Dorrington and Mihaesco give a molecular weight of 920,000
[ ( 5
x
119,000) + (1
x
320,000)], whereas th e data of Plaut an d Tomasi
yield 815,000. Since stoichiometric yields were not obtained in either
study, and variable amounts of peptides were formed, these values must
be considered approximate.
The report by Plaut and Tomasi is particularly encouraging since
F(
c),
p was obtained in substantial yields. The extremely low yield of
F(
c) , in all previous studies has greatly hampered decisive investiga-
tions of this interesting fragment.
c.
d.
3 .
Fragmentation
of
Uncertain Origin
Unusual fragmentation of yM has been described by several authors.
Yakulis
e t
nZ.
(1 96 8) noted progressive formation of Fa b- an d Fc-like
fragments from an isolated yM cold agglutinin after reduction by sodium
borohydride. Some
Fc
fragments were also produced in this way from
rabbit yG and human -,A (Yakulis
e t
al.,
1969). Preincubation with 2-
mercaptoethanol increased the yield of fragments. Albritton et al. (1970)
observed a similar release of Fab- and Fc-like fragments from a Walden-
stroni macroglobulin during incubation with 0.015 M mercaptoethylamine
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88 HENRY METZGER
a t p€l ’7.2 a nd 37°C. The latter authors suggest that previous occult
peptide cleavage led to release of fragments during reduction. Alterna-
tively, disulfide cleavage may have increased the susceptibility of the
molecule to p q t i c hydrolysis by enzymic contaminants. This latter mech-
anism would be consistent with the findings of Rimon et al. (1969) .
They found that incubation of yM proteins (partic ularly certain W alden-
strom macroglobulins) in 5 to 6 M ure a a t p H 5 to 8 led to progressive
release of low molecular weight polypeptides from both p and light
chains. The reaction was not inhibited by iodoacetamide, p-hydroxy-
mercu ribenzoate, or c-aminocaproate ( t h e latter is
a
plasmin inhibitor)
but was diminished
by
exposure to 60°C. They suggest that cleavage
resulted from extraneous proteolytic enzymes. Klein
et
al.
(1967) re-
ported on a 7 s component which almost certainly resulted from con-
tamination of a yM solution with Proteus organisms.
A
well-documented
example of such contaminants splitting
7G
proteins
was
published by
Robert and Bockman (1967).
V.
low
Molecular Weight Macroglobulin-Like Proteins
Immunoglobulins antigenically indistinguishable from yM but having
a
considerably lower molecular weight were first clearly described in
horses (Sa nd or, 1962; Sandor e t
al.,
1964) and then in humans (Roth-
field et al., 1965).
These “7 S-,M” proteins were observed in a variety of disease states:
systemic lupus erythematosis (pa rtic ula rly in male patients ) ( Roth-
field et
al.,
1965; Stobo and Tomasi, 1%7), dysproteinemia (Solo-
mon and Kunkel, 1965; Gleich
e t
al., 1966), hereditary telangiectasia
( Stobo and Tomasi, 196 7), Waldenstrom’s m acroglobulinemia (B ush
et
al.,
19 69 ), various other lympho proliferative disorders
(
Solomon,
1967, 19 69 ), rheum atoid arthritis (Lospalluto, 1968), and several infec-
tious diseases (Klein e t al., 1967) . A similar protein was found among
normal infant an d a dult immunogIobulins and antibodies (H un ter, 1968;
Perchalski
et al.,
1968; Solomon, 1969).
Although antigenically deficient in some cases (e.g., Perchalski, M S ) ,
by and large these low molecular weight yM-like proteins are indis-
tinguishable from the 7s monomers produced by reduction and alkyls-
tion of 19 S ininiunoglobulin. Biosynthetic studies indicate that
7
S yM
is formed directly and at a diflerent rate than 19s 7M (Solomon and
McL aughlin 19 70 ). Th e genetic an d biosynthetic relationship of these
proteins to 19 S YM remains uncertain.
Low molecular weight y M proteins can
be
analyzed for by gel diffu-
sion, using gels sufficiently cross-linked to preven t diffusion of 19
S
yM.
Stobo and Tomasi (1967) employed
4%
acrylamide gels for this purpose,
whereas Solomon (19 69 ) reported the use of 7% agarose gels. T h e possi-
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STRUCTURE
AND FUNCTION Of yh4
MACROGLOBULINS
89
bility that
yM
breakdown products are being detected (Section 1V,C,3)
must always
be
considered (Solomon, 1969).
VI. Functional Properties of Macreglobulint
A.
INTERACTIONITH
ANTIGENS
1 .
Comparative Aspects
of y M
Antigen-Combining Sites
I have already discussed the preliminary sequence data which (with
one possible exception) fail to demonstrate
a
subset of light-chain or
heavy-chain variable regions which is unique to yM proteins. These
data are so preliminary, however, that they cannot provide
a
definitive
answer to the question of whether yM-combining sites are in any way
unique.
The properties of
y M
and yG antibodies produced in response to
immunization with the same antigen have been compared in many
studies. Clearly, if we are concerned about the combining sites, only
those experiments in which the polymeric nature of yM could not in-
fluence the analyses are relevant (Section VI,A,3). By and large, such
studies have dem onstrated t he similarity of yM- an d yG-com bining sites.
Onoue et al. (19%) studying rabbit anti-benzenearsonate, Jaton e t
al.
(19 67) stud ying antibodies specific for uridine, an d A tsumi
et
a,?.(1968)
studying anti-benzylpenicilloyl antibodies fo un d that yM and yG ant ibod-
ies isolated from the same sera had similar binding constants. Voss and
Eisen (1968) and Makela and associates (1967; Makela and Kontiainen,
1969) , on th e other hand , obtained som ewhat lower binding constants for
yM and 7G antibodies isolated from the same bleedings. The specificity
and size of
y
M-combining sites have been investigated and, although in
one s tudy (M.
E.
Kaplan and Kabat, 1966) it was concluded that yM
sites were directed to smaller determinants than yG sites were, other
studies (Groff et al., 1967; Haimovich
et
al., 1969; Moreno and Kabat,
1969) failed
to
reveal significant differences. If we are concerned about
the potentialities inherent in th e yM structure, those studies th at f ail to
demonstrate a difference seem most instructive.
Many factors influence which cells are stimulated, and the immuno-
globulin end product will reflect this process
as
well
as
the potentialities
of the immunoglobulin-combining sites. The subject is considered in
some detail in Section X.
2 .
Valence
of y M
Studies on a Waldenstrom macroglobulin, yMLav,which resembled
“rheumatoid factor,” in possessing yG-binding activity] supported the
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90
HENRY METZGER
assignments for the proteolytic fragments based on structural criteria
( Metzger, 19 67). Those fragments called “ Fa b p” bound to the ant igen,
and the dinieric
‘‘F(ab‘),
p”
fragments not only were twice
as
active
as the monomeric Fab, but fully accounted for all the activity in the
subunits, yM,. Subsequent studies (Stone and Metzger, 1967) showed
that all ten of the YM,,, Fab fragments were active. The failure of the
subunits to bind more than 1 mole of antigen ( y G or Fc 7 ) a t a t ime
(Metzger, 1967; Stone and Metzger, 1967, 1968) was attributed to steric
factors. Such factors did not seem important when the ligand was small.
Thus, a Waldenstrom macroglobulin that bound nitrophenyl derivatives
with high specificity showed th e expected valence of one bind ing site
for each heavy-light-chain pair, regardless of whether proteolytic frag-
ments, subunits, or pentamer were studied ( Ashman and Metzger, 1969)
(F ig . 5 ) .
These results have been complemented by findings on non-Walden-
strom macroglobulins. Chavin and Franklin (1969) studied the yM com-
ponent of
a
mixed cryoglobulin containing both K and h yM molecules
which reacted with yG. They showed tha t a ll the F a b p f ragments were
active despite the fact that only 1 mole of yG or Fc y was bound per
mole yM subunit. Merler et
al.
(1968) studied a human yM antibody
separated from the serum of
a
normal donor who had bcen immunized
with typhoid vaccine. A tetrasaccharide separated by paper chro-
matography from
a
partial hydrolyzate of
Salmonella typhimurium
lipopolysaccharide was employed for equilibrium dialysis. The ex-
periments were technically difficult because it was necessary to read
ligand concentrations
a t
210 mp. and only the protein-free compartment
of the dialysis cells could
be
analyzed. Nevertheless, although the data
showed a moderate degree of scatter, they were consistent with ten
identical combining sites per mole yM. The study by Cooper (1967)
on two cold agglutinins was more indirect but his results supported the
same conclusion. He used subunits from cysteine reduced yM-subunits
in which the consituent polypeptide chains remain covalently linked
(Section IV,A,l ) . Such subunits retained almost all the hemagglutinating
capacity (an d the temp era ture dependence) of the unreduced yM. Con-
ventionally reduced and alkylated subunits were inactive in this study.
Though it was not proven that the agglutination absolutely required
bridging of cells via divalent agglutinins in this instance, it seems rea-
sonable to assume that this was the case. If the subunits are divalent
then the yM pentamer can be assumed to be decavalent.
It
is rather
surprising tha t th ere was only a twofold difference in the hemagglutina-
tion titer for the subunits and th e native yM (se e next sec tion ). Schrohen-
loher et
al.
(1964) and Stone and Metzger (1967) also demonstrated
agglutination by certain yM subunits though the activity of the latter
was reduced 20-100 times compared to the unreduced yM. In these ex-
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STRUCTURE
A N D
FUNCTION
O F -yM
MACROGLOBULINS
25
20
1 5 -
-
2
5
x
>
I O -
0 5 -
I l l ,
, , , I
0 5
10
r
FIG.
5.
Equilibrium dialysis data on a Waldenstroni macroglobulin, yMw.,, which
was fortuitously discovered to bind nitrophenyl derivatives specifically. Binding
was
assayed with
'H-2,4-dinitrophenyl-eNHr-caproic
acid. The data are plotted as
7
(moles hapten bound per mole heavy-light-chain pa ir ) over
c
(moles free hapten) vs. T .
The yM, yM,, F(ab ') ?, and Fab were assumed to have
10,
2,
2,
and
1
heavy-light-
chain pairs (or equivalent), respectively. Calculations were based on molecular
parameters given in
F.
Miller and Metzger (1955a, 1955). (Reproduced from Ash-
man and Metzger, 1969, by kind permission of the publishers.)
periments, however, chain dissociation could have resulted in partial in-
activation consistent with Cooper's results.
Other studies on the valency of yM proteins have led to conclusions
less straightforward than those described above. Results from experi-
ments employing high molecular weight antigens (Franklin
et
al., 1957;
Lindqvist an d Bauer, 1966; Schrohenloher an d Barry, 1968) which showed
a valence only one-half as large as expected can be interpreted as reflect-
ing
steric factors (see a bo ve ) bu t oth er results, from experiments
in
which
low molecular weight ligands were employed (Onoue
et
al., 1965;
Voss
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92 HENRY
METZGER
and Eisen, 1968), are more difficult to understand. A recent paper by
Onoue et al. (1968a) is instructive. They studied yM antibodies from
rabbits immunized with azonaphthalene sulfonate groups attached to
S . typhimurium. Their equilibrium dialysis data showed considerable
heterogeneity: although the “first” few sites could be saturated at
1
to
3
x
1W
M
hapten, further binding was observed even at
2 1
x M
hapten. They interpreted their data as showing equal numbers of weakly
and strongly binding sites and proposed that each
yM
molecule had
five weak and five strong binding sites. This interpretation seems un-
warranted to me and results , I believe, from the tendency of the
Scatchard plot to flatten out disproportionately as the weaker binding
sites of
a
heterogeneous mixture are titrated. Their data when replotted
simply as
c
(t h e fre e ligand concentration) vs.
r
(moles of bound ligand
per mole of protein) fail to show either a break in the binding curve
or a finite valence to which the plot can be extrapolated unambiguously.
The d at a of Voss and Eisen (1968) apparently led to similar ambiguity.
These results, therefore, do not seem to undermine seriously the conclu-
sions described in the first part of this section. (See also Section IX.)
Frank and Humphrey (1968) approached the valency question differ-
ently. They investigated the ability of radiolabeled “subunit~”~f purified
rabbit anti-Forssman antibody to adhere to
a
solid antigen adsorbent.
Forty to fifty per cent of the subunits failed
to
bind. The authors pro-
posed that since only half of the expected amount of binding was ob-
served, there may be only five rather than ten combining sites on each
anti-Forssman molecule, although other explanations for the data were
considered. Their results ar e provocative b u t too indirect to b e con-
sidered definitive.
Unexpected findings were reported by Costea et al. (1966, 1967).
They examined cold agglutinins from normals and patients with in-
fectious mononucleosis, Mycophmu pneumoniae infection, and systemic
lupus erythematosis. Unlike the agglutinins from patients with idiopathic
cold agglutinin disease which contained only K-type l ight chains (and
which were used as controls), the former agglutinins contained both
K and
A
light chains. Surprisingly, th e activity w as completely precipitated
with either anti-K or anti-A serum. This suggested that the vast majority
of agglutinins were mixed ~ , h olecules. When such agglutinins were
reduced and prior to reoxidation precipitated with anti-h or anti-K serum,
activity was recoverable only
if
th e anti-A bu t not if th e anti-K seru m was
used. This result suggests that the original yM agglutinins cons;sted of
a mixture of active
K
subunits and inactive
A
subun its. Th e results resemble
those of Frank and Humphrey but, l ike the latter study, require further
confirmation.
It is clear from a report by Coligan and Bauer (1969) that conven-
These subunits appear to have been half-molecules
of
yM. (Section
IV,A,l ) .
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STRUCTURE AND FUNCTION OF yM MACROGLOBULINS
93
tionally raised yM antibodies are not always composed of a mixture of
active and inactive subunits. In their study more than
90
of
the sub-
units derived from pooled
yM
anti-bovine serum albumin w ere bou nd to
an antigen-cellulose adsorben t. T he stu dy by Merler
et al. (1968)
al-
ready cited makes the same point,
3. Influence
of
Pulyvabncy
The contribution made by the polyvalency
of
yM antibodies to a
variety of secondary antibody-antigen interactions, remains to be as-
sessed. It is intuitively obvious that an antibody with multiple binding
sites will bind m ore firmly tha n a un ivalent or bivalent antibody to a
multivalent antigen. Translating this intuition into precise quantitative
terms has not yet been attempted and may be
of
little value if done
entirely in the abstract. The energetics of binding will be highly de-
pen den t on the num ber an d topology of the antigenic determinants and ,
correspondingly, the distribution of the combining sites in space ( Section
111,AJO; Fig. 3) . There is evidence that the yM molecule has consider-
able flexibility (Sections III,A,S an d 1 0 ) . Th e different conformations
need no t all be energetically equivalent, however, so that the free energy
of binding will
be
a balance between the free energy released by
com bining site-determinant interactions a nd the energy required to
maximize the number of such interactions. In reactions that, in addition,
involve cross-linking
of
translationally independent determinants
(
agglu-
tination) the effect of polyvalency is even more difficult to gauge.
The situation becomes increasingly complex in reactions such as
hemagglutination. Hemagglutination is not simply the cross-linking of
inert particles. With antibody binding, dramatic surface changes take
place which can profoundly influence local charge densities and, in
turn, the relative ease with which two cells can approach (Salsbury
et
al.,
1968; Bangham and Pethica, 1960). These surface changes may
b e influenced by th e local density of t h e antigen-antibody interactions
and this will undoubtedly be influenced by the valence of the antibody.
An experimental analysis of this problem will require systems in
which t he intrinsic combining affinities an d num ber of antibody sites, an d
the number and distribution of antigenic determinants, can all be de-
termined and varied independently. Macroglobulins with homogeneous
combining sites are available. Similarly, techniques for making mixed
pentamers
(
pentamers made
b y
reassociating subunits from active and
inactive
y M ) have been describt,d
(
Kunkcl
et
aE.,
1961;
Jacot-Guillarmod
and Isliker, 1964; Deutsch, 1969; Harboc et al., 1969). Since it should
be possible to conjugate inert particles with statistically defined surfacc
densities
of
antigenic determinants, the problem s e e m experimentally
approachable.
A study in which some of the many variables iiivolved in a precipita-
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94 HENRY METZGER
tion reaction could be controlled has been reported (Stone and Metzger,
19 68 ); it was possible thereby to rationalize m any of th e properties of
that system.
4 . Macroglobulin Antibody-Antigen Interactions:
Secondary Phenomena
Considering the discussions in the previous three sections, it is not
surprising to read in one study (Haimovich and Sela, 1968) that the
relative capacity of
y G
a nd
y M
to inactivate phage varied 1000-fold, de-
pending on the nature of the ant igenic determinant OR the phage.
Similarly, a large number of factors may influence the relative efficiency
of
yM
relative to
y G
antibodies in a variety of secondary phenomena,
such as opsonization and bacterial clearance. Nevertheless, the value of
comparisons of y M vs.
7G
responses is diminished only a little by our
lack of a complete understanding
of
such secondary reactions. W e are,
after all, not only interested in molecular mechanisms but in a descrip-
tion of physiologically important events. The value of many studies
(e.g., Robbins
et
aZ., 1965; Chernokhvostova
et
al., 1969) on
yM
anti-
bodies in a variety of immune responses may, therefore, lie not so much
in what they reveal about the capacities of y M proteins, as in what they
tell
us
about biologically important phenomena. For example, it seems
to me more im portant t o describe wha t fraction of th e total opsonizing
capacity of an antiserum is accounted for by various immunoglobulins
during the course of an immune response than to belabor the opson-
izing capacity of such antibodies per microgram nitrogen in any par-
ticular case. The latter figure by itself adds surprisingly little to our
understanding.
A
useful review on th e beh avior of im munoglobu lins from d ifferent
classes in various assay procedures has be en published (Pike, 19 67 ).
The data can be simply summarized: those subpopulations of yM not
reacting with complement (see below) aside, y M antibodies participate
in all types of imm unological reactions othe r tha n those that involve
fixation to heterologous or homologous tissues.
B. INTERACTIONITH THE COMPLEMENTYSTEM
Complement is a system of eleven serum proteins, an important
(t houg h not exclusive) fun ction of w hich is interaction with cell mem -
branes. The complement system is specifically activated by immuno-
globulins-the antige n-co mbining sites of th e latter determ ining th e
location where activation takes place, The initial step in complement
activation is the interaction of immunoglobulin with the calcium-de-
pendent macromolecular complex C’I
(
Muller-Eberhard, 1968, 1969).
Different immunoglobulins react with Cjl with different efficiencies: y , ,
y z , a n d y 3 react well (Ishizaka et al., 196 7), whereas y 4 and y h react
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STRUCIURE AND FUNCTION OF ’/M MACROGLOBULINS
95
poorly if at all (Ishizaka e t al., 1967; Muller-Eberhard, 1968, 1969).
Macroglobulins were known to activate C’1
for
some time, but i t was
recognized only recently that, in rabbits (Hoyer
et al . ,
1968), mice
(Plotz et al . , 19 68 ), guinea pigs (H ysl op and Matheson, 1967; Linscott
and Hansen, 1969 ) , and humans (Mackenzie et al., 1969), noncom-
plement-fixing
y M
antibodies may also be present.
The mechanisms by which y M a nd yG mediate complement activity
is under active investigation at present. Recent studies indicate that
both the initial activation of C’1
as
well as later steps in the complement
sequence are influenced by the class of immunoglobulin utilized.
1 .
Znitiation
of
Com plem ent Fixation
On cell surfaces a single molecule of
yM
suffices to fix c‘l, whereas
antibody “doublets” are required for fixation by
YG
immunoglobulins
(Humphrey and Dourmashkin, 1965; Borsos and Rapp, 1965a,b). This
difference may be partially explainablc if activation requires saturation
of two sites on a polyvalent C’lq component ( Muller-Eberhard and Cal-
cott, 1 96 6) . If the re is
a
C’l-fixing site for each pair of immunoglobulin
heavy chains, single yM molecules would
be
effective but two yG mole-
cules side by side would be necessary.
Differences in C’1 activation by Y M and yG also become manifest
when the temperature is varied. The
C’1
is efficiently activated by
yG
at both 4” and 3 7”C., whereas
y M
hemolysins are very inefficient at the
lower temperature (Stollar and Sandberg 1966; Colten et al., 1967) .
Using re d cell-hemolysin-C’4 interm ediates
EAC’4), it can also
be
shown that the rate and extent of activation by y M antibody is greater
than when 1G antibody is used
(Col ten e t al., 1969). Whereas yG
antibodies
fix
C’ optimally at equivalence ratios of antigen to anti-
body,
y M
shows maximal activity in antibody excess (Ishizaka
et
al.,
19 68 ). At the optima t h e two classes of antibody were equivalently active
on a molar basis in that study. Other differences between
yM
a nd y C
complexes on cell surfaces and with soluble or insoluble antigens were
noted by the latter workers.
2. Influence on Later Steps in the Complement Sequence
Several complement components appear to attach directly to cell
membranes and not to antibody molecules
(
Muller-Eberhard and Biro,
1963; Miiller-Eberhard e t
al.,
19661 For example, complement conipo-
nents remain attached even when thc hcinolysin is dissociated from the
complex [such as by warming of cold hemolysin-cell complexes ( Harboe,
19 64 )], an d t he lytic secluence may go to completion after removal
of
all detectable antibody ( Muller-Eberhard and Lepow, 1965). Although
these observations at first suggest
a
role for antibody only in the initia-
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96
HENRY METZGER
tion of complement fixation, current investigations suggest that the
immunoglobulin class of the hemolysin may have more complex effects.
Frank and Gaither (1970a) have shown that the temperature-dependent
differences in lytic efficiency described above are demonstrable when
several complement-red cell interm ediates ( EAC’1, EAC’14, EAC’4,
EAC’142, an d EAC“1423) ar e used, thus suggesting th at these inter-
mediates reflect the differences in the hemolysin used for their initiation.
Later intermediates, e.g., EAC’1423567 prepared with yM hemolysin-
sensitized cells lysed equally well at 37” and 4°C. In a second study,
Frank and Gaither ( 1970b) documented substantial differences in rela-
tive titers of whole guinea pig complement and partially purified com-
ponents C’l , C’2, C‘3, C’4, C’5, C’6, C’8, a n d C’9 wh en assayed with
yG- and yM-sensitized erythrocytes. Their results further suggest that
although yM hemolysins may more effectively initiate complement fixa-
tion such sites may utilize complement components more efficiently when
they are initiated by yG immunoglobulins.
It has been observed (Frank et al., 1970) that cells lysed with
yG
antibody may have many more “holes” than those lysed with
yM
anti-
body when excess guinea pig complement is used. Human complement-
induced lysis di d not d emo nstrate such a difference. Th e stage a t which
the hemolysin acts to produce this effect remains uncertain.
The entire complement sequence is required for hemolysis as well
as for other cytolytic and cytocidal effects (Inoue et at., 1968; Inoue and
Nelson, 19 66 ). For other phenom ena, such as precipitation of imm une
complexes (Paul and Benacerraf, 1965), immune adherance ( Nishioka
an d Linscott, 19 63), phagocytosis enhancem ent ( R. A. Nelson, 1965),
chemotaxis (Shin
et
al., 1968; W ard
et al . ,
19 66 ), anaphylatoxin genera-
tion ( Cochrane and Muller-Eberhard, 1968), yM-mediated adhesion to
macrophages
(
Huber
et
al.,
19 68 ), an d virus neutralization (Da niels
et
al.,
1969; Linscott and Levinson, 1969), the participation of only a
limited number
of
complement component is required. The influence of
th e imm unoglobulin class on several of t he early steps in th e com plement
sequence can b e ex pected to have significance for these reactions also.
3 . Inactivation of yM-Initiated Complement Fixation
Complement fixation by yM
is
markedly inhibited after exposing the
immunoglobulin to thiol reagents. Murray e t
al.
(196%) found that
with 0.03 to 0.10M ethanethiol (C,H,SH), yM activation was differ-
entially depressed compared to y G activation. Stollar and Sandberg
( 1966) achieved similar results with mercaptoethanol. Differential loss
of yM complement activation can also be irreversibly affected by heat-
ing (60°C. ) (Murray e t
al.,
1965a) and by exposure to 4 to 5 A4 urea
at 37°C. (Cunniff e t al., 1968) .
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STRUCTURE
AND
FUNCTION
OF yM MACROGLOBULINS
97
c. INTERACTIONS WITH OTHER PROTEINS
Weak associations between macroglobulins and other proteins have
been described.
A
series of eight specimens for patients with Walden-
strom’s macroglobulinemia showed such weak interactions with serum
albumin, leading to unusual inimunoelectrophoretic effects
(
Hartmann
e t al., 1966). Association between yG and y M from normal individuals
has also been observed (Filitti-Wurmser
et al.,
196 6), bu t neither reac-
tion has been extensively characterized.
A more complete study of albumin binding to yA myeloma proteins
and Waldenstrom macroglobulins was published
by
Mannik
( 1967b).
He found complexes that would only dissociate after reaction with re-
ducing reagents ( 2-mercaptoethanol was used) but not by denaturing
reagents such as
5
M guanidine hydrochloride. References to earlier
works on the association of immunoglobulins to various serum proteins
can be found in Manniks paper .
The significance of these interactions remains obscure.
D.
INTERACTIONS
ITH
CELLS
Specific receptor sites on macrop hages a nd other leukocytes for the
Fc region of yG immunoglobulins have been described (Uhr, 1965;
Berken an d Benacerraf, 1966; Rabinovitch, 19 67 ). A sep ara te site, sensi-
tive to trypsin and directed toward the third component
of
complement
can also cause binding of immunoglobulins to leukocytes by way of
antigen-antibody-complement complexes ( “immune adherance”) ( D. S.
Nelson,
1963;
Lay and Nussenzweig, 1968; Huber
et al., 1968).
Macro-
globulin-antibody complexes can be bound by this mechanism ( Huber
e t al., 1968; Henson, 1969) though the reaction is weaker and, curiously,
may
be
enhance d by th e presence of uncom plexed
yG
( H u b e r
et
al.,
1968).
A distinctive receptor for yM immunoglobulins on mouse macro-
phages which requires Ca++ons and which is not destroyed by trypsin
has been investigated by Lay an d Nussenzweig (19 69 ). Th e binding is
reversed if Ca++ s decreased by dilution or chelation. It is unclear at
present whether the receptor sites for guinea pig yM studied by Del
Guercio et al. (1969) are similar to the latter sites. A useful discussion
of the difficulties that may have led to the early failure to recognize
yM
receptor sites may
be
found in the paper by Lay and Nussenzweig
The l imited data on this subject are no reflcction of its importance.
Phagocytosis of immune complexes is undoubtedly a major defense
mechanism, and specific cell receptors for immunoglobulins can
be
ex-
pected to play a critical role in this process.
( 1969) .
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98 HENRY METZGER
VII.
Genetic
Basis of
Macrog lo bu l in Structure
A.
IZVIDENCE
FOR SUBCLASSES
I have already cited certain studies which suggest that human yM
immunoglobulins may be divisible into subclasses. The relationship be-
tween th e classes suggested by Filitti-Wurm ser et al. (19 64) on the basis
of sedimentation rates (Section III,A,I ), by Davie and Osterland ( 1968)
on the basis of carbohydrate content ( Section III ,B ,2) , an d b y Mackenzie
et al. (1969) on the basis of ability to fix complement (Section VI,B) is
completely unknown. The possibility that low molecular weight macro-
globulin-like proteins and hexameric
yM
molecules result from unique
p-chain loci must also be considered (Section 111,AJO and
V ) .
Additional evidence for subclasses comes from studies by Harboe
et al. (1965) and by Franklin and Frangione (1967, 1968). Harboe et al.
immunized rabbits with 7M immunoglobulins isolated from patients with
Waldenstrom’s macroglobulinemia. One such antiserum when partially
absorbed with normal human serum reacted with 9 out of
22
sera from
patients with Waldenstriim’s macroglobulinemia and 5 of 5 normal
serums. Franklin and Frangione (1967) similarly obtained a rabbit
antiserum which when absorbed with an antigenically deficient serum
reacted with one-third of forty macroglobulins. The reaction was not
related to the solubility properties, electrophoretic mobility, or light-
chain typ e of t h e protein. Im porta ntly, each of twenty-six no rmal sera
reacted with the absorbed antiserum. In subsequent work, Franklin and
Frangione (1968) showed that the Fab p fragments carry the determi-
nant in question an d that tryptic pep tide maps of the p chains contained
one or two distinctive spots correlating with the serological differences.
In addition, these maps suggested that the seropositive and seronegative
groups might each be divisible into two further subtypes.
Interpretation of these data must be provisional. An important ques-
tion is whether these differences are related to variations in amino acid
sequence (a n d , hence, to the p-chain ge ne s) or only to differences in
carbohydrate (a nd , hence, of undetermined orig in). Although F ranklin
and Frangione (1968) have obtained peptide map differences, this does
not rule out carbohydrate differences which might
( a )
influence the
mobility and R , of peptides directly or ( b ) affect the site
of
t ryptic
hydrolysis. That each
of
twenty-six normal sera
was
reactive with the
antiserum is strong evidence against the variations being due to allelic
polymorphism. Similarly, that both noncomplement-fixing and com-
plement-fixing yM proteins can be found in the sera of inbred mice
(Plotz et al., 1968)
is
not explainable on the basis of simple allelism.
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STRUCTURE
AXD
FUNCTION OF yM
MACROGLOBULINS
99
B. ALLELICMARKERS
Macroglobulins can potentially show allelic variability due to differ-
ences in light chains, heavy chains, or both. Aniong human proteins only
light-chain I n V variants have
so
far been dvtected. The K-type y M pro-
teins of
an
individual will evidence the
same
InV determinant(
s )
as the
other K-type immunoglobulins. Unlike the situation with y2 heavy chains
-where combination of that subclass of heavy chains with
K
chains may
result in inefficient expression
of
the InV determinant
(
Steinberg and
Rostenberg, 1969), p chains do not appear to affect the IiiV marker
appreciably. The light-chain allotypes of rabbits are similarly easily
recognized on
yM
proteins.
Allelic markers referrable to chains have so far been found only on
rabbit proteins. Here the
(Y, p,
and
y
chains appear to have a common
marker: the operationally defined a locus with alleles Aal, Aa2, Aa3
(Todd, 1963; Feinstein, 1963; Lichter, 1967; Pernis et al., 1967). Quantita-
tive precipitin data suggest that the allotype marker is identical in each
of these heavy-chain classes (Pernis
et al.,
1967) (cf. Segre
et al.,
1969).
Initial studies, furthermore, indicate that there are shared amino ter-
minal sequences in
(Y
and y chains which correlate with the allotype
(
Wilkinson, 1969a,b). Since the structural data indicate that the common
regions of these heavy chains are coded for by distinctive genes, the
allotypic data which indicate shared loci suggest that the heavy chains
may be coded for by two separate genes. Furthermore,
a
genetic marker
(shared or not) in the variable region would seem to place important
constraints on theories of antibody variability. These matters are dis-
cussed at length in Colin
(1968),
Edelman and
Gall
(1969), Metzger
( 1970), and Hood and Talmadge ( 1970).
Intraspecific differences in antigenic determinants, distinct from the
a and
b
loci determinants, have been reported for rabbit
yM
(Kelus and
Gell, 1965; Sell, 1966; Kelus, 1 367).
The
determinants (dubbed Ms 1,
Ms 2, .
. . )
may require the presence of certain a and b alleles in order
to be expressed (Kelus, 1967).
A full analysis of these markers has not
yet been published.
C. IDIOTYPICARKERS
As with other immunoglobulins, individual yM proteins may be used
to
preparc antisera which will
bc
directed to structures more or less
unique to the immunogen. They were first described for
y M
proteins by
Habich (1953), and more recent references may be found in Harboe
et al. (1969). Intcrestingly, some of these idiotypic determinants may
be specific for proteins having a common function. For example, Williams
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100 HENRY METZGER
et al. (1968) described certain antisera that demonstrate Specihcity for
groups
of yM cold agglutinins but not for yM immunoglobulins lacking
such activity. One cannot rule out the possibility that these determinants
may relate to minor subclasses or alleles, but this seems unlikely (Wil-
liams
e t
d . 1968). It is interesting in this respect that thirteen of fourteen
cold agglutinins reacted positively with the antiserum described by
Franklin and Frangione ( above) (Franklin, 1969).
VIII. Biosyn thes i s and Me tabo l i sm of M a c r o g l o b u l i n s
A.
BIOSYNTHESIS
1 .
Cellular
Origins
Cells producing 19S-yM immunoglobulin are not distinguishable as
a group from cells producing other immunoglobulins, by either con-
ventional or electron microscopy (Harris et al., 1970). Both
YG
and yM
immunoglobulins are produced by two types of cells, lymphocytic and
plasmacytic, each of these types showing considerable morphological
variability. In the rabbit, 80-85% of
yM
plaque-producing cells were
of the plasmacytic
form.
Substantially similar results were obtained with
mouse cells. The paper by Harris et al. (1969) describes some of the
elegant techniques used
to
isolate and examine functionally relevant
cells.
One study (Nossal et al., 1964) suggests that single antibody-pro-
ducing cells may switch from macroglobulin to yG synthesis, but one
would like to see more extensive supportive data. Immunofluorescent
data that show essentially all cells as producing a single class of heavy
chain (Cebra
et nl.,
1966)
do
not necessarily conflict with Nossal
et
al.’s
results
if
the switchover time is short. More extensive data on cells in
tissue culture indicate that single cells can produce both
yM
and other
immunoglobulins (Fahey and Feingold, 1967; Takahashi
et al.,
1968)
but one must be cautious about extrapolating from these data to physio-
logical situations. As discussed elsewhere ( Metzger, 1970), the data on
the homogeneity of immunoglobulin receptors on individual presump-
tive antibody precursor cells are conflicting.
2. Assembly
The first study on the cellular assembly
of
yM molecules has just
appeared. Parkhouse and Askonas (1969) studied the incorporation of
tritiated leucine into y M proteins by cell suspensions of the Balb/c
mouse tumor, MOPC 104E. The cells secreted
y M
and light chains in a
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STRUCTURE: AKD FUNCTION OF y M MACROGLOBULINS
101
1:2 weight ratio with a lag period of
20
to 30 minutes. Within the
cells, a 7 S component (presum ably yM ,) accumulated a nd even after
3
hours of incubation only traces
of
completed 19
S
molecules were
detected in the cell lysates. On the other hand, essentially all of
the secreted macroglobulin was of the 19 S variety. The authors conclude
tha t “T he processes of polym erization an d secretion ar e . . . intimately
related, affording a mechanism fo r th e selective secretion of th e larg e
molcules.”
As indicated in Section VI,A,Z, the light chains of individual
yM
molecules h ave been said to b e mixed K a nd h (Cos tea e t
ul.,
1966, 19 67 ),
suggesting postsecretion assembly of subun its or, alternatively, homo-
geneous with respect to light-chain allelic markers ( Schmale
et
al.,
1969) ,
suggesting assembly prior to secretion. Data on allelic markers on the
p-chains sup port the latter result (Pern is
e t
al., 1967).
B. DISTRIBUTION
Macroglobulins are distributed predominantly in the intravaseular
pool both in rabbits (Ta liaferro an d Talmadge, 1956) an d humans
(Cohen and Freeman, 1960; Barth
et
al., 1W ) .Unlike yG antibodies,
no extensive maternal-fetal transp ort of yM immunoglobulins takes place.
In a stud y with iodinated proteins in h umans, Gitlin
et al.
(1964 ) demon-
strated some transfer of yM-the fe tal blood level reach ing
~ 1 0 %
f the
maternal concen tration, bu t most feta l yM (m ea n concentration 0.10
mg. /ml . ) appears to be accounted for by local synthesis (Van Furth
et nl., 196 5). Th ere is no correlation between yM (o r r A ) levels in the
maternal and fetal sera (Stiehm a nd Fu den berg , 19M; Johansson a nd
Berg, 1967).
C.
RATES
OF
SYNTHESIS
ND CATABOLISM
An extensive review of immunoglobulin metabolism has appeared
recently (Waldmann and Strober, 1969) and I will, therefore, summarize
the data on yM only briefly.
In humans, synthesis of yM generally reaches substantial levels around
th e twentieth week of gestation (V an Fu rth et at., 1965; Toivanen
et
al.,
1969). In hum ans, 4.5-6.9 mg. of yM ar e synthesized pe r kilogram p er
day-equivalent to 2 x l o L i molecules for the “standard 70-kg. in-
dividual. This is only about one-twentieth the number of yG molecules
produced per day. On the other hand, the fractional catabolic rate
(see
Waldmann and Strober, 1969) is 2-3 times that of yG, and this rate is
unaffected by the serum concentration over a greater than 1000-fold
range (Barth et
al.,
19 64 ). As with other imm unoglobulins, the sites of
yM catabolism are uncertain. The lower synthetic rate and higher cata-
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102 HENRY METZGER
bolic rate account
for
the relatively low level of
yM
in normal human
serums: 0.8-0.9 mg./ml.
Many other details regarding the metabolism of
y M
and other
immunoglobulins in both normal individuals and in patients with
a
variety of disease states will be found in Waldmann and Strober’s
review,
IX. Macroglobulin-like Proteins f r om Nonm ammalian Species4
Cyclostomes and higher forms synthesize high molecular weight anti-
bodies which resemble mammalian immunoglobulins. Some properties
of th ese proteins ar e listed in Tab le
VII.
Not shown in the table are the
recen t electron-microscopic results of Feinstein a nd M unn
(
1969) which
indicate that dogfish and chicken high molecular weight antibodies
have a configuration very similar to
y M
from several mammalian species.
At the risk of being overconservative, however, it seems prudent to
reserve judgement
on
the extent to which these nonmammalian high
molecular weight an tibodies ar e homologous to mamm alian yM (i.e.,
are directly derived from a common ancestral heavy-chain gene).
For
example, it is impossible to exclude rigorously that they may be more
closely related to mammalian
yA.
Where measurements have been made, the heavy chains
of
these
proteins appear to have
a
molecular weight of +70,000, similar to the
most frequently observed value for mammalian p chains.
The
molecular
weight data for the whole immunoglobulins as well as the electron-
microscopic data cited above indicate that many of these proteins are
likely to have a pentameric structure. Recent evidence for a hexameric
“yM” from the frog Xenopus levis (Parkhouse
et
al., 1970) (Fig. 4) and
micrographs
of
the high moleclar weight antibodies of carp which appe ar
tetrameric (Shelton, 1970) (F ig. 6 ) show,
on
the other hand, that
each system deserves independent exploration.
Many nonmammalian sera show a prominent 7 S immunoglobulin
component. I n th e elasmobranchs this 7 s protein is difficult to dis-
tinguish from the
7
S subunits derived by reduction from the 18S com-
ponent ( Marchalonis and Edelman, 1965; Clem a nd Small, 196 7). Th e
proteins have similar antigenic properties, and their coiistituent poly-
peptide chains have similar disc electrophoretic mobilities, molecular
weights, amino acid compositions, and tryptic peptide maps. One dis-
tinguishing feature observed by CIem and Small (1967) remains un-
explained. They found that, whereas the 18 S antibodies lost their ag-
glutinating capacity upon reduction, th e 7 S antibodies did not. Possibly,
’Two useful
reviews
appeared
too
late
to
be discussed in this article:
Clem
and
Leslie, 1970;
Grey,
1969~ .
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TABLE
V I I
N O N M A M M A L I A N
ACROGLOBULIN-LIKE
ROTEINS~J
Cyclostomes Elasmohranchs (3) Teleosts 4) A
Pacific
hagfish (1). Lamprey
( 2 )
Pro1:erties High
1.0w
High Low High
I.ow
High
Low
Hi
I. Structural
Sedimentation rate > 2 8 S
14
S 6 . 6 s -19s -7 s 16 S 6 . 4
S
1 8 s
~ o i .t . x 1 0 - 5
K.R.
- h-.R. 1.0 8.7 f O . 6 1 .6 f O . 0 9 9 . 0 1 . 2 X . R
1 42 - N.R. N.R. 1.34 1.38 1.3 8 1.66 N.R.
. 1 %
c 280mc
Carbohydrate(%
)
3 .4
-
N.R.
N.R. 3.7
3 . 5
“Hi&”
“ L o w ”
ihexose) (hexose) (hexose) (tot ,
Heavy-chain
mol.
w t . X 10-4 N.R. - N.R. 7 .0 7 .1 7 .1 7 .0 4 0 7 .2 f
11. Functional
10.8
Light-chain mol. wt. X 10-4 N.R. - K.R. 2
5
2.2 2 . 2 2 2 2 2 2 . 0 f
Valence K.R. ~ K.R. K.R. 5-10 1-2
5-10
1-2
a Numbers in parentheses indicate the following referenres:
(1) Thoenes and Hildemann (1970).
(2) Marchalonis and Edelman (1968).
(3)
Marchalonis and Edelman (1965): Clem and Small (1967);
Voss
et a t . (1969); Iilapper et nl. (1970); Clem el n l . (196
(4 )
Clem and Small (1870).
(5) Marchalonis a nd Edelman (1966).
(6)
Leslie and Clem
(1969);
Gallagher and
Voss (1969):
Orlans
et
n l . (1961).
a “High” an d “Lou.“ indicate high and low molecular weights, respectively. X.R.
=
not, reported.
c A
low molerular neight immrinoglobulin has not been reported for this speries.
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104 HENRY
METZGER
the 18S antibody sites had a rather low intrinsic binding affinity which
in the polymer was amplified because of polyvalency.
Th e relat ionship between th e 7 s and the 1 8 s immunoglobul ins i s
still uncertain as
is
true for the 7S-yM-like proteins in mammals (Sec-
tion V ) . Du rin g a n immu ne response the ratio of 7 S to 18
S
activity may
increase (C lem an d Small, 1 96 7), suggesting th e possibility th at the 7
S
and 18s may be more distinctive immunoglobulins than present data
would suggest.
Although the situation in teleosts remains unclear (Clem and Small,
1970), in higher forms (amphibia, etc.), 7 S components distinctively
different from yM proteins are discernible (Marchalonis and Edelman,
1966).
Data on the valence of nonmammalian immunoglobulins are in a
confusing sta te of affairs a t this writing-another prop erty which the y
apparently sh are with mam malian yM Th e recently published stud y of
Voss et al. (1969) on lemon shark an ti-dinitropheny l antibodies concludes
that there is one effective site per 7 s unit an d five per pentamer, bu t
close examination of their data suggests that their extrapolations may
be somewhat arbitrary. Clem a nd Small (1968, 1970) studied the valence
of immunoglobulins of the giant grouper ( a marine teleost) , but a
definite value cannot yet
be
assigned. The extreme heterogeneity (and
rath er low average binding constants
)
evidenced in the published binding
data suggests that a useful approach may
be
to isolate a more limited,
tightly binding se t of molecules-for exam ple, by frac tion al pre cip ita-
t ion. The 7 s components hav e sometimes been reported to have bi-
valent properties (Suran
et
d. , 1967; Clem e t al., 1967; Clem an d Small,
1967) and other times not to have (Voss et al., 1969). Larger amounts
of material and s tudy of Fab and F(ab’) , fragments (Klapper et
al.,
1970) may help to resolve the issue.
X. Role o f M a c r o g l o b u l i n s i n t h e Im m u n e Response
So
far, I have concentrated on the structure of yM macroglobulins,
on their interaction with antigen, the complement system, and with
certain cells. Ultimately, it is desirable to incorporate these structural-
functional relationships into a coherent scheme for the role-particularly
for an y special role-that yM antibod ies perform in th e imm une response.
Antibodies appear to function in two important ways in the immune
response. First, cell-bound antibodies serve as antigen receptors on
those cells tha t ar e th e imm ediate precursors of antibody-pro ducing
cells, those cells that mediate cellular immune reactions, and those
antigen-sensitive “helper” cells that appear to facilitate the productive
interaction between antigen and antibody precursor cells ( Metzger,
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STRUCTURE AND FUNCTION OF
yM
MACROGLOBULINS
105
1970). Second, sec rete d antibody participates in the control
of
further
specific antibody production apparently b y influencing the availability of
antigenic determinants to the cell-bound receptors
(
Uhr and Moller ,
1968).
In this section
I
shall consider some of the data which suggest
that yM antibodies may subserve these functions in unique ways.
A. MACROGLOBULINSs ANTIGENRECEPTORS
There is l i t t le information with respect to the nature of the antigen
receptor on helper cells or on those cells that mediate cellular immunity.
The weight of evidence is in favor of their being immunoglobulins
(
Metzger, 19 70 ). O ne stu dy implicates conventional light chains on
these receptors (Greaves et
al.,
19 69 ), bu t there is no information
as
to
the type ( if any) of heavy chain involved. On the other hand, there is
substantial evidence that immunoglobulins of all classes are involved
on those cells which will produce antibodies or daughter cells which
will. Macroglobulins can be envisioned as playing a special role as re-
ceptors on such cells for one or more of the following reasons: ( I t he
combining sites of yM antibodies may exhibit certain specificities not
present among immunoglobulins belonging to other classes;
( 2 )
the
polyvalency of yM receptors may influence the ease with which the
cells bearing such receptors are triggered; and 3 ) yM-bearing cells
may have uniq ue functions.
The first point has already been discussed (Section VI,A,l ). There
are no substantial structural or functional data which suggest that
yM antibodies have a unique set of specificities.
Data with respect to the second point have been presented by
Makela and associates ( 1967; Makela and Kontiainen,
1969).
Reason-
ing that a multivalent receptor would react more strongly than a bi-
valent receptor with multivalent antigens, they studied the influence of
antigenic valency on th e class of antibody p roduced . Their initial results
suggest that, indeed, a multivalent antigen markedly enhances the yM
response compared to that observed with a paucivalent antigen.
If
t he
yM-bearing cells were partly being triggered on the basis of the
re-
ceptor valency rather than simply on the basis of the intrinsic binding
affinity of receptor sites, then the yM antibody sites might have a lower
intrinsic binding constant than the simultaneously produced
rG
anti-
bodies. Some experimental confirmation of this prediction has been
obtained (Makela
et
al., 1967; Makela an d Kontiainen, 196 9). Th e
data suggest that early in an immune response yM receptor-bearing cells
might
be
stimulated prior to significant
cell
selection by antigen (see
Siskind an d Benacerraf, 1969). Whether this accounts for the frequently
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106 HENRY METZCER
observed fact that y M antibodies tend to be preferentially synthesized in
the early stages of the immune response is unccrtain but it is certainly
likely that it contributes to the phenomenon.
Several groups (N ao r and Sulizeanu, 1967, 1969; Hu m ph rey an d
Keller, 1970; Ada and Byrt, 1969; Ada et ul., 1969; Byrt and Ada, 1969)
have recently reported on the distribution and effect of highly radioactive
antigens added to unprimed lymphoid cells. Both Humphrey and
Keller and Ada and associates found that with such “hot” antigens the
imm une response could be inhibited-presumably b y killing th e cells
to which th e radioactive antigen ha d b ound. Significant here is tha t such
uptake has so far been inhibited only by anti-light-chain and anti-p-chain
sera (Ada et
al.,
19 69 ). It is not known wh ethe r helper cells or anti-
body precursor cells or both are being affecte d in this stu dy , bu t i t
suggests that there are certain lymphoid cclls bearing yM receptors
on their surface that may be critical to the production of serum anti-
bodies. There is evidence that yM antibodies were the first to evolve
(Section IX ) and the y are usually the first to app ear in ontogeny (G oo d
an d P apermaster, 1964; Sterzl and Silverstein, 196 7). It would not be
surprising if the m atu re imm une response continued to reflect th e special
evolutionary position of yM imm unoglobulins.
B.
MACROGLOBULINSN
THE
CONTROLF ANTIBODYYNTHESIS
T h e specific role of secreted y M antibodics in the control of antibody
production is complicated and there is much conflicting information
(U h r and Moller, 1 968 ). A recent s tudy by Henry an d Jerne (19 68)
gave some clear-cut and provocative results. They administered purified
yM and yG antibodies before or shortly after immunization with a
homologous antigen. The yG antibodies suppressed the subsequent im-
mune response, whereas the yM antibodies markedly enhanced it .
Mixtures of
yG
and y M antibodies gave the expected algebraic sum
of
th e individual effects. Fu rthe r studies are required to know just how
general these effects are and the mechanism of action. Henry and Jerne
suggest tha t 7 S antibodies may cover imm unogenic determ inants, whereas
cytophillic Y M antibodies, by binding antigen to macrophages, m ay
promote contact between antigenic determinants and relevant receptors
on lymphocytes.
XI. Prospects
The weight of evidence now favors a molecular model of yM con-
sisting of a circular ( usually pentam eric
)
array of equivalent symmetrical,
four-chained subunits, each containing two equivalent (though not
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STRUCTURE
AND FUNCTION OF YM MACROGLOBULINS
107
always simultaneously available ) antigen combining sites. The evidence
against such a model has been cited but, in my view, it is unconvincing.
Those proposing more exotic models have yet to prove their case.
Those pursuing the primary sequence of the
p
chain will settle the
remaining ambiguities about the size of these proteins, and further molec-
ular weight studies on garden-variety macroglobulins seem to me a
bad investment of effort at this juncture. Though occasional crystalline
yM
proteins have been described,
I
am not aware of any promising
FIG. 6. Electron micrograph of tetrameric inrinunoglobulin isolated by Drs.
M . Sniith and
E .
Shelton from carp. (The micrograph was kindly provided
by
Dr. E. Shelton.)
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108
HENRY METZGER
diffraction studies and we may, for some time to come, have to satisfy
ourselves with extrapolations from X-ray patterns
of
yG immunoglobu-
lins and with the more indirect, but increasingly sophisticated, optical
methods of conformational analysis.
The most interesting problems remain: How do these molecules func-
tion-both as cell receptors and as effectors for antigen disposal? If they
were the most primitively arising immunoglobulins, what came before
them? Does their present role still reflect their putative unique place
in the origins of the immune response? Providing the chemical answers
to these questions will require the combined talents
of
protein chemists
and cellular biologists.
ACKNOWLEDGMENTS
I am indebted to many colleagues who sent me preprints of completed work
and work still in progress. The Journal of Biological Chemistry, Immunology, and
Nature kindly perm itted reproduction of published figures, Mrs. Pe arl Goldhagen’s
attention to detail was an invaluable aid in assembling this manuscript.
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TRANSPLANTATION ANTIGENS 119
enzymes (Davies, 196613, 1967; Nathenson and Davies, 1966; Summerell
an d Davies, 1969; Shim ada a nd Nathenson, 19 69 ). Similarly, proteolytic
digestion of human spleen cells (Sanderson and Batchelor,
1968;
Sander-
son, 1968 ) a n d of cultured leukocytes derived from donors with lym phoid
malignancies
(D.
L. Mann
e t
al., 1968, 1969 a,b) resulted in t h e extraction
of solubilized alloantigens.
The longtime and widespread use of sonic energy to liberate water-
soluble substances from intracellular, intraorganelle, and membranous
locations on bacterial and animal cells (Grabar, 1953) led to the early
application of this method in an attempt to solubilize transplantation
antigens. Billingham et
aZ.
( 1956b) applied probe-mediated, high-
frequency, high-intensity ultrasound
(20
kc./sec., 60
W.
)
and detec ted
only small amounts of soluble, immunogenic mouse transplantation
antigen, whereas Haughton (1964) found that antigen was released and
then rapidly inactivated after exposure to ultrasound. O n the o ther h an d
application of low-frequency, diaphragm-mediated sonic energy ( 9-10
kc./sec.; 15 W.) made it possible to obtain reasonable amounts of water-
soluble transplantation antigens from mouse spleen cells ( Kahan, 1964
19 65 ), from guinea pi g spleen (K ah an and Reisfeld, 1967) an d sarcoma
cells (Kahan et al., 1969 ), from dog spleen cells (D ag he r et al., 1967) ,
from human spleen cells (Kahan
et
al.,
1968b), and from cultured
leukocytes derived from normal donors (Reisfeld
et
al., 1970a).
1 1
Extraction and Solubilization
of
Transplantation Antigens
The whole concept of solubilization of histocompatibility antigens
from their site on the cell membrane surface is based upon the assump-
tions that 1 ) antigen molecules can be isolated independent of the
membrane structure, (2) the solubilized product represents the antigen
molecule with its antigenic determinants more or less in the same form
as on the cell membrane, an d 3 ) here ar e no im mun ologically significant
intermediate linkages between solubilized antigens a nd other constituents
of the membrane.
Definitions vary as to what constitutes a solubilized antigen. Many
investigators consider an antigen to be in a soluble form
if
it does not
sediment when subjected to centrifugation at lOO,OOOg, i.e., is free of any
visible cell membrane fragments. This concept is questionable since
Rapaport et al. (1965) found that antigen which did not sediment at
100,OOOg did contain membrane fragments, as determined by ultra-
structural studies of sediments obtained from these “soluble” antigens
following centrifugation at
200,OOOg.
Some of the definitions as to what
constitutes a ‘‘soluble” and “stabilized antigen preparation are not
too
meaningful. Davies ( 1968) has distinguished “solubilized from “stabi-
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120
R.
A.
REISFELD AND B.
D.
KAHAN
lized” antigens on the basis that the latter are chemically complex poly-
molecular lipoprotein complexes which do not sediment in the presence
of detergents during short-term high-speed centrifugation and which are
immunogenic but not separable molecular entities. Soluble antigens as
defined by Davies (1968) can be fractionated into molecular entities
carrying different antigenic determ inants an d possessing b inding capacity
for cytotoxic antibodies. Blandamer e t al.
(1969)
defined “as an accept-
able dem onstration of solubility that the antigenic material shou ld be
able to pass throu gh
a
gel filtration column and not be eluted with the
void volume.” To use the behavior of antigenic material on Sephadex
columns is not entirely acceptable since it
is
known that these m aterials
can easily aggregate and thus be excluded. Most properly, the solubility
of a substance should be defined by its characteristic solubility in any
given solvent th at d oes not d en atu re it. Since most an tigenic preparations
isolated thus f ar ar e complex mixtures of proteins an d conjug ated proteins
subjec ted to intric ate protein-protein interactions, classic solubility
characteristics, such as those used
to
define protein purity, are hardly
applicable. Thus, at present, it is most useful to define solubility in
practical terms, by the absence of membrane fragments detectable
by
ultrastructural methods and by the applicability
of
methods, such as ion-
exchange chromatography and gel electrophoresis, which resolve antigen
preparations without serious loss of biological activity due to insolubility
a t varying pro tein concentrations in a num ber of aque ous solvents.
The question of solubility may be of critical importance during the
evaluation of purification especially in the assessment of yield. It is
conceivable th at t h e loss of antigenic units may reflect th e precipitation
of insoluble antigenic materials contaminating the “soluble” preparations.
A
useful and practical criterian of solubility consists of prolonged dialysis
of th e antigenic preparations against distilled w ater followed b y extensive
ultracen trifugation a t 130,OOOg witho ut serious loss of an tigenic activity
of the supernatant.
Numerous attempts have been made to solubilize transplantation
antigens from cell membranes with detergent and organic solvent treat-
ments, with proteolytic digestion, and by application of sonic energy.
A. DETERGENTXTRACTION
1.
Triton
and Potassium
Cholate
Kandutsch (1960 ) found that
5%
Triton X-100 was able to extract an
antigenic material from particulate fractions
of
t he
A
strain mouse
ascites tumor Sarcoma I. This material was almost completely insoluble
in water in the absence of detergent and a t pH values in the region of
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TRANSPLANTATION ANTIGENS
121
neutrality. A molecular weight of less than 200,000
was
stipula ted since
the sedimentation coefficient (s2,,,) in the presence of Triton was 2.65
( Kandutsch, 1 96 3). T he yield of the whole Triton extract comprised from
30 to 40% f the weigh t of th e freeze-dried cell particulate fraction. T rea t-
ment of this extract with 0.1 M sodium phosp hate buffer, p H 6.9 an d
with 0.14
M
saline resulted in a preparation ( 5 4 by weight of the
lyophilized particulate fraction) which did not sediment when centri-
fug ed a t 100,000g for 30 minutes. This extract, which was soluble in dilute
salt solution when treated with snake venom, of which the active com-
ponent was thought to
be
phospholyase A, could be readily dissolved into
a slightly opalescent solution in 0.1 A 2 tris buffer, p H 7.9. How ever, w hen
centrifuged at 100,OOOg for
2
hours, approximately
50%
of this material
was sedimented. The snake venom-treated preparation showed only one
major compon ent by moving bound ary electrophoresis ( p H ran ge 6.6-
9.0), but ultracentrifugal analysis revealed this material to be quite poly-
disperse suggesting th at th e a ntige n was e ither a collection of polymeric
units or still contained small cell m emb rane fragme nts. It is qu ite possible
that actua l electrophoretic heterogeneity of this complex substance was
masked by the alteration of its overall charge due
to
the presence
of
lysophosphatide g roups following enzym atic trea tm en t, A similar problem
arises when other detergents, e.g., sodium dodecyl sulfate, are applied in
electrophoretic characterization of antigen preparation. It is also feasible
tha t th e polydispersity observed was d u e to extensive chem ical complexity
rather than to polymeric forms of
a
single substance. T he enzyme-treated
material seemed to be composed of lipo- and glycoproteins since analyses
showed the following: nitrogen, 8.91%;hexose, 2.38%; hosphorus, 0.97%;
lipid, 33.7%. In an at tempt to elucidate the chemical nature of the anti-
genic determinant, Kandutsch (
1963)
extracted the antigen with chloro-
form-methanol and found that most of the antigenic activity was
destroyed as was the case when more purified preparations were treated
with 0.004 M sodium-m-periodate. Kandutsch and Stimpfling (
1966)
also attempted enzyme treatments of thcir antigenic preparations. Thus
trypsin [enz ym e/sub strate ratio ( E / S ) = 1:5; 2 hours at room tempera-
ture] did not markedly affect antigenic activity and resulted in little
fragme ntation since most of the digest was excluded by Sephad ex G-200.
Pronase ( E / S = 1 : l O ; 17 hours at room tem pera ture) caused 75%of the
material to be included in Sephadex G-200. However, this included
material had little or
no
antigenic activity.
In a recent pape r, Hilgert e t al. (1969) evaluated the effectiveness of
three detergents-Triton X-100, Triton X-114, and potassium cholate-to
solubilize H-2 antigen from
a
particulate fraction
of
Sarcoma I. T h e
"Cr-cytotoxicity assay was used
to
measure antigenic activities. Triton
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122
R. A. REISFELD
AND
B. D.
KAHAN
X-100 was fo un d to b e least suitable since, although th e de terg en t cxtract
contained 50%of t he initial activity, abou t two -thirds of it was lost wh en
acetone and e ther had to
be
used to remove the detergent. Triton X-114
was more advantageous to use than X-100 since it could be used for a
shorter period of time ( 2 hours compared to 1 0 day s) an d at lower
concentrations
( 3
p1. of 20% Tr iton X-114 as compared to 10
pl,
of 20%
Triton X-100 per milligram of p ro te in ). Most of th e Triton X-114 could
be removed with ether without any detectable loss of antigenic activity
after saturation of the water p ha se with am monium sulfate. However, this
prep aration was insoluble in th e absence of a dete rgen t in dilute salt
solutions a t p H 7. Gel filtration on either agarose or S eph adex G-200 in
the presence of
1
riton X-100 did not result in any significant concen-
tration of antigenic activity.
Attempts to solubilize the antigen with potassium cholate (varying
KC1 concentration from 0.14 to
3
M while cholate was varied from 0.1
to
1 )
esulted in approximately the same yield ( 2 0 5 0 % of the initial
antigen activity) with th e same increase in specific activity (tw o- to four-
fold) as did solubilization with Triton X-114. The advantage claimed
was that exposure to organic solvents was not necessary to remove the
detergent. Fractional ammonium sulfate precipitation did not result in
any significant separation of five
H-2
specificities; however, much of the
protein was not retained on Sephadex G-200 and was either aggregated
or had a relatively high molecular weight. The specific activity of the
retained fraction was not significantly higher than that of the excluded
fraction.
2. Deorycholate
Metzgar et
al. (
1968 ) employed (0.5W)deoxycholate to disrupt h um an
tissue culture cells ( K B and W2-38 cell lines or chimpanzee lymph node
cells) and were able to solubilize the
4"
an d 4b antigenic determinants
of the human HL-A system. After the addition of deoxycholate, deoxy-
ribonuclease (2.2 mg ./lOg cells) was ad de d and th e mixture incubated
for 15 minutes a t 37°C.; MgCl, (0.08
M )
was then added and th e mixture
incubated for an additional
15
minutes. The suspension was adjusted to
0.4
M
MgCl, concentration to precipitate deoxycholate and centrifuged
at 100,OOOg for 1 hour; th e sup erna tant was dialyzed against two changes
of phosphate-buffered saline overnight, recentrifuged at 100,OOOg for 30
minutes, an d stored at -75°C. This antigenic preparation specifically
inhibited agglutination of leukocytes, mixed agglutinations, and cyto-
toxicity reactions and induced the accelerated rejection of donor skin
grafts, Metzgar et al. (19 68 ) also extracted lymph nodes from chimpan zee
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TRANSPLANTATION
ANTIGENS
123
donors by the same procedure and employed these extracts to test for
inhibition of agglutination by h um an isoantibodies. Th e chimp anzee
lymph node extract inhibited the reactions of one human antiserum with
a panel of cells from four human donors. It was concluded that, although
the amount of isoantigen recovered could not
be
accurately measured,
from 20 to 30%of the activity of the intact cells was recovered by this
extraction procedure. Although other investigators
(
Brent et al., 196%)
failed to obtain a ntigen ic activity afte r treatm ent of m ouse tissues with
various detergents, Metzgar e t al. (1968) contend that their short-term
( 30-minute) treatment with deoxycholate followed by its quick removal
contributed to their success. Apparently, long-term (
24-hour)
detergent
treatment also failed in their hands to yield active antigen preparations
from mouse tissues.
Bruning et
al.
(1964, 1968) were able to use deoxycholate to extract
HC-B antigens, the products of a less important genetic locus than the
major HL-A locus, from the cell membrane sediments of placental tissue.
This procedure involved the treatment of placental tissue particulate
fraction with 0.5%deoxycholate for an unspecified period of time and
then centrifugation of the extract at 100,OOOg for 2 hours followed by
dialysis of the supernatant against distilled water ( 3
x
24 hours ) and
lyophilization. From
150
to
200
gm. of placental tissue was obtained
200-500 mg. of material which contained
5”
and 5” antigens as determined
by the neutralizing activity of these antigens in agglutination inhibition
assays.
Gel filtration of the extract
on
Sephadex G-200 showed that antigenic
activity could only be recovered from a fraction that eluted in the void
volume even when the extract was previously treated with enzymes such
as
chymotrypsin, lysozyme, or phospholipase
A.
It is, of course, possible
that residual deoxycholate may have prevented enzymatic action.
3.
Decyl and Dodecyl Sulfate Extraction
Manson and Palm
(1968)
obtained
a
solubilized material by extrac-
tion of murine microsomal lipoproteins with sodium decyl and dodecyl
sulfate. This material specifically inhibited alloantibody and remained dis-
persed after removal of t he detergent. Sed imentation constants ascertained
ranged from 15 to 55 and permit only speculation concerning the actual
moleculsr weight of these preparations which apparently were composed
of highly complex lipoproteins.
Manson et
al.
( 1963) also obtained microsomal lipoprotein particulates
by exposing mouse tumor cells to 1500 p i . or 15 minutes followed by
decompression in a nitrogen bomb and ultracentrifugation of thc sus-
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124 R. A. REISFELD AND B . D. W A N
pension. This particulate lipoprotein material was shown to inhibit
hemagglutinating antibody and to contain homograft-sensitizing activity
at dose levels (intraperitoneal) from 25 to
50
pg.
B. EXTRACTION
ITH
ORGANICOLVENTS
Another extraction method to obtain solubilized histocompatibility
antigens involved th e use of orga nic solvents. Morton (1950) demon-
strated that intracellular enzymes could
be
both released and solubilized
by extraction of various cell particulate fractions. Butanol was found to
be a most effective solubilizing ag en t for enzymes including phosphatases,
peptidases, dehydrogenases, esterases, and transaminases. In contrast,
some of the intercellular enzymes studied were destroyed by extraction
with papain, autolysis, lipases, or trypsin. Because of its relatively
low
solubility, n-butanol saturates aqueous solutions without causing any
serious loss of activity when ex tractions are carried ou t at 0" to -2°C.
It is postulated that butanol competes most effectively for the polar side
chains of proteins with the alcohol displacing the lipids and, thus,
causin g dissociation of lipoproteins an d protein-protein complexes.
Morton (1950) also obtained some evidence that phosphate esters are
preferentially extracted by butanol thus destroying th e structu ral in-
tegrity
of
cell membrane-bound lipids resulting in the solubilization of
membrane proteins.
Kandutsch
(1960)
used butanol to solubilize the antigenic activity
of a membranous fraction obtained from water-lysed Sarcoma I mouse
ascites cells. The butanol extract of the cell particulate fraction was
centrifuged at 105,OOOg
(1
hour) at room temperature, the supernatant
poured into 10 volumes of cold acetone, and the resultant precipitate
washed with ether and dried in
vacuo.
Saline suspensions of this material
enhanced the survival of grafts. Manson and Palm (1968) liberated from
40 to 75 of th e H-2 an d non-H-2 an tigenic activity of m icrosomal lipo-
proteins with butanol. The lipid content of the solubilized material
was
reduced from
42
to 28%bu t still existed in a highly agg regated form w ith
a particle weight estimated from an
s
value of 56 to
be
as hig h as
6 x
l o G.
This material was ineffective in inducing accelerated graft rejection when
injected intraperitoneally but caused an accelerated allograft response
when injected subcutaneousIy in Freund's adjuvant. The butanol-solu-
bilized material also elicited formation a nd a nam nestic rise of H-2 anti-
body and inhibited donor-specific hemagglutinins and cytotoxins. Harris
et
al.
(1968) used Triton X-100 treatment and butanol extraction to
solubilize antigen from cell membrane fragments
of
rabbit lymph nodes
an d spleens. A nalyses of these cxtracts by sucrose density gradient centrif-
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TRANSPLANTATION
ANTIGENS
125
Ligation and diethylaminoethyl (D E A E ) -cellulose chromatography indi-
cated them to be of highly complex chemical nature.
A
number of investigators have tried to extract soluble histocompati-
bility antigens with detergents and organic solvents from
a
variety of
animal and human tissues. However, the overall results have, in retro-
spect, not been encouraging. Although many of the extracted materials
show ed goo d an tigenic activity an d specificity an d often imm unogenicity,
they were generally found to be such extremely complex mixtures that
there was little or no effort made to purify them further and to charac-
terize them by physicochcmical methods. Furthermore, there is well-
founded and reasonable doubt that these materials instead of being
really truly water-soluble were often composed of small membrane
fragments in a colloidal suspension. Although it may yet be feasible to
obtain truly soluble antigens with new detergents, at present i t appears
that this approach has not been too rewarding.
C . ANTIGENEXTRACTION
Y
PROTEOLYSIS
1 . Autolysis
Digestion of membrane fragments of mouse spleen and tumor cells
and human lymphoid cells with proteolytic enzymes has been used by
several investigators to solubilize histocompatibility antigens.
In order to solubilize murine histocompatibility antigens, Nathenson
and Davies (1966) washed niesenteric lymphoid, thymus, and spleen
cells for
20
minutes with
0.8
and
0.7%
saline. T h e extract was centrifuged
a t 6OOg for 15 minutes and the combined supernatants centrifuged at
105,000g for 90 minutes. The sediment contained at best 70% of the
activity of the original cells. This crude insoluble lipoprotein fraction
was suspended in tris-HC1 buffer, pH 7.4, and decreased the antigenic
activity. The autolysate was centrifuged at 105,OOOg for 1 hour, an d the
sup ern ata nt was tested for antigenicity by its capacity to inh ibit cyto-
toxic antibody. T he ab solute amou nt of solubilized activity thu s obtained
did not exceed
20%
of that in the insoluble starting material, i.e.,
14%
of
the activity of the original cells. Although antigens could be solubilized
from normal C3H mouse lymphoid tissue by autolysis, this method failed
when applied to
BP
8 ascites tumor cells. In this case, the crude mem-
brane fraction
was
incubated with k i n for 1 hour at
37°C.
a t a n E/S
rat io of 1
16.
Ficin digestion for more tha n
1
hoiw d estroyed the antigenic
activity of the membrane fraction.
The crude ficin containing at least eighteen proteolytic enzymes was
partially purified by gel filtration on Sephadex
G-75.
Only thc eluate of
the inclitded volumc
was
uscd for digestion of th e crudc, mem brane f r x -
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126
R. A.
REISFELD
AND B.
D.
KAHAN
tion. The enzymatic digest was centrifuged
105,OOOg
for
1
hour and
passed over Sephadex G-75. Antigenic activity was found only in the
included volume in accord with previous knowledge of the behavior of
solubilized murine alloantigens upon gel filtration ( Kahan, 1965).
Autolytically solubilized antigen was passed over Sep hadex G-75 an d
all antigenic activity was found in the excluded fraction in the void
volume. Gel filtration of this material on Sephadex G-200 resulted in
removal of some inactive protein and in a fourfold increase of activity.
Stepwise elution chromatography of these antigen preparations on
DEAE-Sephadex w ith increasing sodium chloride concentration (0.1-
0.15 M ) resolved three fractions, two of which showed a twenty-fold
increase in activity. Antigens thus prepared
(1
could be lyophilized,
(2 ) were soluble in aqueous solvents at pH 6.5 at a concentration of
1 mg./ml., (3 ) were heat-labile ( 2 minutes at 6 Oo C . ) , a nd ( 4 ) were
stable at
37°C.
only in the pH range 6-9. Ultrasonication
(30
seconds;
60 W.;
20 kc.) resulted in a 20% loss in activity, and exposure to urea
( 6 M ) a t 37°C. irreversibly inactivated the antigen within
20
minutes.
Analyses of this antigenic material showed protein
60-64%
by weight,
amino sugar 8.5%,and hexose 7%.Phosphate content was less than 0.2%
and lipid content was not determined. All three DEAE-Sephadex peaks
contained the same antigenic specificities, i.e., there was no apparent
separation of H-2 specificities. Fr om 600 mice, approximately 1 gm. crude
membrane fraction was obtained, 90 mg. of which was eluted from
Sephadex G-75 (one- to twofold increase on specific activity), and the
two most active Sephadex fractions contained 5 mg. with a twenty-fold
increase in activity. From th e Sephadex elution patterns (G -7 5 an d
G-200) the molecular weight was estimated to be between 75,000 and
200,000.
Davies (1967) passed an autolysate of murine spleen mem brane frag-
ments over a column of Bio-Gel
P-300
and tested various fractions dif-
ferentiated from a ra the r diffuse elution pat tern for inhibition of im mu ne
cytolysis. The portion of the eluate that was partially in the included
volume of the column and comprised a relatively large portion of the
total protein effluent was further purified on DEAE-Sephadex using a
straight-line salt gradient (0.05M tr is , p H 7.4, plus NaCl to get a
molarity from
0.1
t o 0.35). Biological activity was localized in the middle
of a broad protein (O.D. 280) distribution pattern. Electrophoresis of
this material on
a
sheet of acrylamide gel
( p H 7.2;
300
mA.; 15
V./em.;
15.5
hours ) revealed a relatively d i h s e electrophoretic zone
situated between the a- and p-globulins of an adjacent reference pattern
of normal mouse serum. Another adjacent strip containing 20 pg. of
antigen was cut into sections and eluted with water and activity was
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128
R.
A. REISPELD
AND B.
D.
KAHAN
norm al murine ( A N ) cells an d from BP 8 tumor cells. Soluble extracts
of the latter were found to induce consistently the formation of hemag-
glutinating antibody and, in
two
cases, the induction
of
cytotoxic anti-
body as well as the accelerated rejection of allogeneic skin grafts. Ion-
exchange chromatography of soluble antigen extracts o n DEAE-cellulose
and subsequent analyses of column eluates by acrylamide gel electro-
phoresis revealed highly complex patterns especially in fractions with
antigenic activity.
No
attempts were made to purify or characterize these
antigen fractions.
2. Antigen Extraction
by
Papain Digestion
Human isoantigens were solubilized from spleens by autolysis and
by papain treatment of crude membrane fractions obtained by hypotonic
salt extraction
(
Davies
et
al., 19 68 a,b ). Isoantigens were evaluated by
their inhibitory capabilities for cytotoxic antisera and for platelet com-
plement fixation. Crude salt-extracted membrane fractions were allowed
to stand 2.5 hours at 37”C., centrifuged at 120,OOOg for 90 minutes, and
the supernatants were found to contain the autolysed alloantigens. The
sediment, resuspended in 0.05 M tris buffer, p H 8, was incubated with
p a p a i n ( E / S
=
1:l.W) in the presence of 0.35
m M
cysteine for 45, 90,
and 180 minutes. Enzyme digestion was most effective at 180 minutes
and was stopped by the addition of iodoacetate. Papain was removed by
passage of the material over Sephadex G-75 columns. These antigens
were fractionated either by autolysis, papain digestion, or both proce-
dures combined, by gel filtration on Bio-Gel P-300, and by ion exchange
chrom atography on DEA E-cellulose or DEA E-Sephadex columns. The
point was m ade th at there are “good” and “bad” spleens, and only the
former could be efficiently extracted by papain digestion. In the mouse,
it was found that the efficiency of solubilization of isoantigens varies
from strain to strain. Much was made of the observation that these rela-
tively crude solubilized extracts containing either H-2 or HL-A allo-
antigens behav ed similarly on DEAE-cellulose. How ever, in bo th cases,
relatively poor resolution was obtained.
The
antigens also showed similar
profiles upon gel filtration on Sephadex G-200. From this and from the
fact that th e same methods we re able to extract HL-A an d H-2 isoantigens
from lymphocyte membranes, Davies
et al.
(1967) deduced that mouse
and human histocompatibility antigens were homologous, i.e., molecules
with closely similar composition.
Although this is certainly a possibility the antigens isolated thus far
are chemically highly complex and heterogeneous and arguments based
solely on these data are not too convincing. Wliethcr or not histoconi-
patibility antigens of different species are homologous in a manner
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TRANSPLANTATION ANTIGENS
129
analogous to immunoglobulins will become evident only after thorough
chemical analyses are performed on highly purified antigen preparations.
Boyle (
1970),
analyzing homogenates obtained from human cadaver
spIeens, found the major HL-A activity associated with a microsomal
sediment which could be further fractionated by sucrose density gradient
centrifugation, the activity being associated with two of three major
components. Antigens were assayed by either inhibition of leukagglutina-
tion or lymphocytotoxicity. The sedinirnt could
be
partially solubilized
a t p H 10 or in
6
M urea which resulted, h ow eve r, in a 90% loss of anti-
genic activity. Phospholipase A from Crotulus adamantus did not solu-
bilize antigenic activity to any significant extent. Digestion of the sedi-
ments with papain
( E / S
=
1:lOO)
for
1
hour at
37°C.
in th e presence of
0.005
M
cysteine at p H 7.2 solubilized H L- A antigenic activity. How ever,
the sole criteria of solubility applied are the absence of either visible
sediment after ultracentrifugation at 105,OOOg for 30 minutes or the lack
of protein stainable material at the origin of
a
cellulose acetate strip
following electrophoresis. By this method, one cathodic and four anodic
components could
be
detected. These conditions of papain digestion
yield solubilization of 20 to 50% of HL-A alloantigens containing 30-402
of th e protein present in the cell mem brane sediment. Additional purifica-
tion was found by gel filtration on Sephadex G-150 where several anti-
genic specificities were detected in a relatively broad zone located
between the excluded an d included vo lu~ nes.H igh specificity was claimed
based solely on the observations that a single specificity not detectable on
the donors’ spleen cells was also absent from the Sephadex eluate which
was found to contain the four antigenic specificities detected on the
donor cells. Specificity ratios, antigen concentration, and cytotoxicity
units at which tests were carried out are not supplied making it d%cult
to evaluate this study.
Sanderson and Batchelor (1968) used insoluable papain since they
found considerable nonspecific inhibition of cytotoxicity even when the
enzyme had b een inactivated b y iodoacetate. Antigens wer e evaluated b y
their capacity to inhibit specific cytotoxic antibodies. Sanderson (
1968
)
emphasized the importance of the specificity of this inhibition since a
variety of nonspecific substances could cause inhibition
of
cytotoxic anti-
bodies. The specificity was evaluated by the specificity ratio. This ratio
was expressed by the amount of antigen needed to inhibit donor-positive
antiserum vs. th e amount tha t can h e used without getting any inhibi-
tion of donor-negative antiserum. The higher the ratio the greater the
specificity which is probably the best index of the degree of alloantigen
purification attained.
Autolysis-solubilized material gave a low specificity ratio ( S R ) ( 1-5)
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130
R . A. REISFELD AND
B. D.
KAHAN
but contained all the specificities possessed by the spleen donor. Papain-
solubilized material gave a h igher SR (a s high a s 12 0) but di d not possess
all the specificities present
on
the donor's peripheral lymphocytes.
Sanderson (19 68 ) pointed ou t that, in the case of antigens solubilized by
autolysis, serologically active substances were bound together with non-
specific inhibitors, and, hence, no real purification of antigenic specifi-
cities was achiev ed. Estimates of th e mo lecular weigh t of papain-
solubilized HL-A antigens were approximately 45,000 based on elution
patterns of calibrated Sephadex G-200 columns. On the other hand,
human alloantigens solubilized by autolysis were claimed to have molec-
ular weights as high as 200,000,
Shimada and Nathenson ( 1969) recently described some chemical
properties of solubilized H-2 alloantigens w ith H-Zb an d
H-2a
genotypes.
The authors started with 4000 spleens of either CS7B1/6 or DBA/2 mice.
The crude particulate fraction was found to contain 354% f the protein of
th e original hom ogenate bu t 901% of th e original a lloantigenical activity,
thus providing a 2.5-fold purification. Alloantigens were solubilized from
this crude particulate fraction by autolysis (1hour a t 37°C.) followed
by papain digestion (E/S
=
1:38) fo r 1 hour at 37OC. The enzyme
digest was centri fug ed a t an ave rage of 78,OOOg for 2 hours-a spe ed
insufficient to sedimen t mem brane particles-and the sup ern ata nt was
subsequently fractionated
by
gel filtration, ion-exchange chromatography,
and acrylamide gel electrophoresis.
The papain procedure was found better in this study than the autol-
ysis method, both will respect to yield and reproducibility. Yields of
alloantigens obtained by autolysis of cell membrane fragments at 37°C.
were maximal at 1 hour and did not change during an additional 8-hour
incubation. Antigen yields obtained by autolysis were found to
be
only
S -l m of th at obtain ed by papain digestion-a claim wh ich differs from
previous observations both by Davies (1967) and by Nathenson and
Davies (1966) who noted that considerably higher antigen yields were
obtained by autolysis. Apparently, autolysis solubilizes murine allo-
antigens to
a
much lesser extent than papain; however, essentially all the
antigenic specificities of the cell donor are present in the autolysate. It
is, indeed, possible that autolysis, at 37"C., although found less efficient
by Shimada and Nathenson ( 1969), solubilizes mainly specificities
located in areas easily accessible to enzymes (cathepsins) present in
finite concentration, On the other hand, papain added in excess, is more
efficient an d probab ly attacks areas on the membrane not accessible
to
cathepsins an d, in the process, inactivates som e of th e specificities which
it solubilizes as well as some antigen which it cannot remove from the
membrane.
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TRANSPLANTATION ANTIGENS 131
Experiments designcd to determine
E / S
ratios and incubation times
showed that incubation periods in excess of 1 hour resulted in
loss
of
antigenic activity as did
E / S
ratios lowcr than 1:30.
An
E / S
ratio of
1:11
was shown to cause approximatcly a 33% loss in specific activity (Shimada
and Nathenson,
1969).
Fractional ammonium sulfate precipitation, gel filtration, and ion-
exchange chromatography were applied in an effort to purify papain-
solubilized H-2” an d H-2a alloantigens. Partially purified antigens of each
genotype resolved into three components each following acrylamide gel
electrophoresis a t p H 9.4. Th rce electrophoretic components within each
genotype were claimed to be separated by the same electrophoretic pro-
cedure apparently containing the same antigenic profile and amino acid
composition. Only small differences in arginine and glutamic acid were
observed between H-2h an d H-2d alloantigens. H owever, th e significance
of these amino acid compositions is difficult to evaluate since only a
single analysis was performed and, thus, it is impossible to determine the
stan da rd error and th e significance of other ap pa ren t amino acid d if-
ferences ( Shimada and Nathenson, 1969).
Th e molecular weights of murine H -2” an d H-2d alloantigens were
estimated by gel filtration and sucrose gradient centrifugation. It was
claimed that antigens from both genotypes could exist either as 65,000-
75,000 or as 40 000molecular weight fragments. Should these estimations
prove correct, then one can either attribute this variation in size to
variable fragmentation by papain or to selective aggregation following
papain fragmentation.
Yields
of
crude, papain-solubilized alloantigen varied with antigenic
specificities and ranged from
15.9
( H-2.5) to 2.6% ( H-2.31), whereas
yields of autolytically solubilized an tigen rang ed from 1.77 to 0.93%. In
the best case (H -2 .5 ), there was usually from one purification s tep to
the next, a two- to eightfold increase in specific activity and about a
700-fold increase in activity concomitant with an overall loss during
purification of approximately
85%
of the alloantigenic activity originally
present in the crude cell membrane extract.
D.
L.
Mann et al.
(1968)
described the isolation of alloantigens from
continuous cultures derived from the lymphoid tissue of human donors
with lymphoid malignancies. Crude papain ( E/S = 2: 1) was used for
1
hour at 37°C. to solubilize alloantigens from frozen cells washed with
isotonic and hypotonic salt solutions. It is of interest that with this
extremely high
E / S
ratio of 2 : l (even though crude papain was used)
from 40 to 50%of the total isoantigenic activity was recovered. However,
in a subsequent paper
D.
L. Mann
et al.
(1969b) showed that with
crystalline papain ( E / S = 0.5 unit mg. pr ot ei n) only 20% of th e m em-
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132
R. A. REISFELD AND
B.
D.
M A N
brane activity could be recovered. Ammonium sulfate-precipitated (0.75
saturation) protein was centrifuged at 40,OOOg for SO minutes and the
dialyzed supernatant further fractionated on Sephadex G-150. The ma-
jority of the HL-A-3 alloantigenic activity of the cell line RAJI was
found in the included fraction, whereas in a subsequent study using a
different cell line (R -42 65 ) the only activity tested w ith a monospecific
antiserum (H L -A -2 ) was found mainly in th e excluded volume (D. L.
Mann et al., 196913). H um an a n d m ouse cell mem brane extracts obtained
by papain digestion were admixed and placed on a Sephadex G-150
column. From the relatively broad but superimposable activity curves
obtained, it was concluded that papain-solubilized fragments may have
similar molecular weights
(
50,000-70,000) an d similar structures in b oth
species. Since these are rather complex and, possibly, in part aggregated
materials, this statement seems to be an oversimplification, especially
since it is well established tha t substances wh ich ap pe ar to ha ve identical
K n
values on Sephadex have more often than not decided chemical
differences.
I n their most rec en t stud y of cell lines RAJI an d R-4265, D. L. Mann
et
al.
(1969b) supply figures for the recovery of HL-A-3 alloantigenic
specificity (R A JI ) indicating that from
3.3
gm. of cells (3.8
x
lo9 cells)
they obtain
0.09
mg. protein with a twenty-fivefold increase in activity
per milligram, representing approximately 3.5%of the activity of the
initial membrane extracts, Since amounts of protein and total units of
activity recovered for LA-2, 4d and 6b specificities (R-4265) are not
supplied (on ly units of activity per milligram p rotein ar e give n), it is
difficult to compare the antigen yields from these two cell lines.
Antigenic activity was determined by the capacity of alloantigens to
inhibit cytotoxic effects of specific alloantisera and complement against
"Cr-labeled targ et cells. An antiserum dilution which would cause lysis
resulting in 60-70% release of ra dio label from these ta rg et cells was
designated as the lytic end point, i.e., 100% ysis. T he reciprocal of th e
dilution of the alloantigen which caused 50% nhibition of lysis, i.e., 30-
35%51Cr release, was used to express units of alloantigenic activity. Thus
in the most purified antigen preparation, approximately 0.05 pg. antigen
could reduce th e lysis of 100,000 target cells from 70 to 35%.
a.
Lability
of
Antigenic Determinants.
Papain which can readily
solubilize some of th e alloantigenic specificities of lymph ocyte m em bran es
can apparently also selectively destroy antigenic specificities. Sanderson
(19 68) fou nd some antigenic specificities wer e not stable to pap ain an d
were, in fact, even destroyed in the insoluble membrane fractions
re-
maining after pap ain digestion. Shimada and Nathenson (19 69) found
that recovery of antigenic activity varied among alloantigens with dif-
ferent genotype and among antigenic specificities within each of the
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TRANSPLANTATION ANTIGENS 133
genotypes. Thus only about half the recovery was obtained with H-2d
as com pared to H-2h alloantigens. Fro m H-2h antigens the H-2.5 activity
was recovered in yields almost two magnitudes higher than the H-2.2
activity, and from H-2I antigens the recovery of H-2.4,10,13 activity was
more than a magnitude greater than that of H-2.31 activity. It is unclear
whether these differences were properties of the antigen or of the anti-
body-detection system.
Shimada an d Nathenson (19 69) also foun d th at only abo ut 18% of
H-2.5 activity present in the crude particulate fraction was solubilized
(60% of activity remaining on the m em bra ne s), whereas H-2.2 was only
solubilized to the extent of 5.5%with 67% activity remaining in th e c rud e
particulate fraction. It seems th at in ea ch case ab ou t 25% activity was
either lost or destroyed. Moreover, H-2.2 which was solubilizcd to a much
lesser extent was not found associated with other specificities such as
H-2 .33 a n d H-2.28. Som e specificities, i.e., H-2.22, were destroyed on t h e
membranes and not solubilized at all,
So
far as th e H-2d alloantigens we re
concerned , H-2.4,10,13 we re solubilized to t he ex tent of 16% with only
22% activity remaining in th e c rud e particulate fraction. In this case it
seems that about two-thirds of activity was not accounted for, i.e., was
destroyed by papain even while remaining in the particulate fraction.
Only 3.6% of th e H-2.31 ac tivity was solubilized, b u t in this case 74% of
activity remained on the me mb rane fragm ents. I t is of c onsiderable
interest that the particulate fraction once digested with papain could
be redigested and additional alloantigens
(from 2 to 10%)could be
solubilized. However, no activity remained in the particulate fraction
following this treatment. This antigenic activity which amounted often
to
as
much
as
70-75% of the initial activity was completely destroyed by
papain, i.e., this enzyme could not remove the vast majority of antigens
on the cell membrane fragments, but apparently it could readily destroy
their serological activity once the easily solubilized antigens
(15%
f the
total) were removed. In this regard, D . L. Mann et
d.
1969b) also
reported poor recoveries of HL-A alloantigenic specificities 4a, 4d,
6b
following papain digestion of cell membranes derived from human
lymphoid cells in culture.
3 .
Antigen Extraction
with Trypsin
Edidin (1 96 7) prepared a stable insoluble stroma by extracting pools
of mouse embryo lymph nodes, spleen, liver, and thymus with hypertonic
salt solutions containing exhylenediaminetetraacetate
( EDTA) (
1.14
M
NaCI, 0.02% ED T A ). This insoluble material was treatcd with trypsin
( p H 7.4) in th e presence of 5 M urea, followed by extraction with 0.14 M
phosphate-buffered saline containing EDTA, centrifugation at
80006,
and finally phenol (88%) xtraction of the resultant supernate which was
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134
R.
A. FIEISFELD AND
8 . D.
KAHAN
lyophilized. Gel filtration of this material on Bio-Gel
P-2
yielded soluble
antigens which specifically inhibited anti-H-2 cytotoxic alloantisera.
These antigenic materials could be further purified by isolation from
specific antigen-antibody complexes dissociated by dilute acid treatment.
Antigens thus prepared seem to be peptides or glycopeptides which were
retarded on Sephadex G-25 and G-10 and were readily dialyzable and
soluble in 5% richloroacetic acid ( TCA). The isolated antigen prepara-
tions were chemically complex and seemed to consist of many antigenic
determinants linked on a single molecule.
The use of a highly specific enzyme such as trypsin has as yet not
been fully investigated and may possibly prove to be a useful method to
solubilize alloantigens from cell membrane surfaces or from insoluble
fragments bearing antigenic determinants.
4 .
Limitations
of
Enzymatic Solubilization Methods
The autolytic method first described by Nathenson and Davies (1966)
has now been found, generally less efficient and less reproducible than
the “papain method (Shimada and Nathenson, 1969; Sanderson, 1968).
Autolysis, generally ascribed to the action of cathepsins seems to attack
the membranes more slowly than papain and “solubilize” large molecular
weight
(
l o 6 )
fragments containing all the antigenic specificities present
on the peripheral lymphocytes of the cell donor. In most cases, digestion
periods can safely be extended from 1 to 8 hours without either decreas-
ing the yield. However, the yields of antigen liberated by autolysis from
the cell membranes vary considerably. Nathenson and Davies (1966)
obtained a cell extract which contained 70%of the activity of that of the
original cells, 20% of which, i.e., 14%of the activity of the original cells,
was solubilized. Recently, however, Shimada and Nathenson ( 1969)
found that autolysis liberates only from
1
to 2% of the activity present
in the crude cell extract. It seems logical that extended periods of auto-
lytic digestions can safely be used since no enzyme
is
added, and the
native cathepsins, which are present in finite amounts, are simply allowed
to act on the cell membranes, However, human alloantigens solubilized
by autolysis have much less antigenic specificity
(SR
= ”1) than papain-
solubilized antigens ( S R = ~ 1 2 0 )Sanderson, 1968). The large molecu-
lar weight of the solubilized material and the finding that it almost always
elutes in the void volume of Sephadex G-200 (Shimada and Nathenson,
1967;
Sanderson,
1968)
while containing all detectable antigenic specifi-
cities of the donor cells leads one to suspect that autolysis may possibly
“solubilize” small membrane fragments. This assumption gains further
support if one realizes that all of the autolytically “solubilized prepara-
tions were always subjected to short-term centrifugations never exceeding
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TRANSPLANTATION ANTIGENS 135
120,OOOg. It would be of interest, in view of the findings
of
Rapaport
et
al.
(1965), to examine the ultrastructure of these preparations or to
expose them to prolonged centrifugation periods at 200,OOOg and above.
Papain has been used in a variety of ways in an attempt to solubilize
with the highest possible yields of the alloantigens present on the
cell
membrane. Various enzyme substrate ratios have been used, varying
from E / S ratios of 2 : l (cru de enzyme used) to 1:150. However , there
is general agreement that incubation in excess of 1 hour results in in-
activation of both human and murine alloantigens and that certain
alloantigens either cannot be solubilized by papain or are obtained in
very poor yield. In fact, some alloantigens are not solubilized at all but
are destroyed on t he membrane.
It
is of interest that, although
a
second
treatment with papain solubilizes additional small amounts of antigens,
it destroys all the remaining antigenic activity which
is
often as much as
60-70%
of that present on the original cell extract.
Up to 700-fold purifications have been achieved for murine antigens
(
Shimada an d Nathenson, 196 9), unfortunately without any serious
att em pt to evalua te specificity ratios of these purified m aterials. C ritical
evaluations of purification schemes of human alloantigens can claim
maxinial specificity ratios of 2120 (Sanderson, 1968). The problems of
papain solubilization of histocompatibility antigens can
be
illustrated by
a close look at the d ata of Shimada an d Nathenson (19 69) . Fro m 4000
mouse spleens, which these investigators calculated to contain
240
mg.
antigen, papain solubilizes about 130-fold as much protein as antigen
present (5310 mg.) and subsequent purification at best resulted in
three
electrophoretic fractions containing
a
combined total of 1.43 mg. T he
activity units per milligram protein were increased 700-fold but at the
same time 98%of the activity units of the crude extracts were lost during
the process of purification.
D. L. Mann
et
al. (1969b) have shown that papain causes from
15
to 20% nonspecific inhibition of cytotoxic activity. These authors, f ur ther -
more, claimed that the nonspecific effects were eliminated by a
1:2
dilution of their test antigen. Sanderson (1968), however, was suffi-
ciently worried about the nonspecific cytotoxicity of papain to consider
mandatory the removal of the enzyme by chromatography following
digestion or to use insoluble papain which could be easily removed by
centrifugation.
D.
SOLUBILIZATION
Y
SONICATION
Exposure of mouse and human tissues to sound and ultrasound has
been widely used in an attempt to liberate soluble histocompatibility
antigens. The activity of the liberated antigens depends upon the condi-
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136
R. A. REISFELD AND B .
D.
KAHAN
tions of sonication. Substantial quantities of potent antigens are released
from cells and their membranes by exposure to low-frequency, low-
intensity sound; however, only small quantities of less active material are
liberated by high-frequency ultrasound. Thus, Billingham
e t
al. ( 1956b)
could detect only small amounts of immunogenic mouse transplantation
antigen in the soluble fraction and, similarly, Haughton (1964) found
that antigen was released but rapidly inactivated after exposure to ultra-
sound generated a t
20
kc./second with
a
60-W. probe.
O n th e other hand , in accord with a large bod y of evidence ( Ch am bers
and Florsdorf, 1936; Haas, 1943; Stumpf
et
aZ., 1946; Pappenheimer an d
Hend ree, 1949; Hogebroom an d Schneider, 195 0), sonic energy m ediated
by a diaphragm of 9 to
10
kc./second liberates active, water-soluble com-
ponents from intracellular, intraorganelle, or membranous locations. The
disparity between the ability of these
two
forms of sound to liberate
active histocompatibility antigens is probably related to the more pro-
nounced oxidative, bond-breaking, and depolymerizing effects of the
probe-generated 20-kc./second ultrasound with its propensity toward
the development of local heating and the generation of eddy currents.
T he re is app arently a relatively narrow region of intensity in which
sonic energy causes solubilization without inactivation of the histo-
compatibility antigens. The effects of sonic
(
<
6,000 cycles/second
)
and of ultrasonic ( >16,000 cycles/second) energy have be en critically
reviewed (Grabar, 1953;
Hughes and Nyborg, 1962). Exposure to sound
breaks up animal and bacterial cells as well as molecules in solution
by
the generation of heat, by oxidative effects, by mechanical effects includ-
ing an agitation effect analogous to foaming, and
by
a frictional effect.
The most important effect, however, is gaseous cavitation with rapid
expansion and violent collapse of the dissolved air within the fluid. The
extent
of
these effects depends upon the intensity and frequency of the
applied sou nd a n d upon th e physical state of th e exposed m aterial.
Exposure to low-intensity sound ( 9-10 kc./second) liberated water-
soluble histocompatibility antigens from m ouse spleen, lung, kidney, a n d
liver cells (Kahan, 1964a,b, 1965; Zajtchuk et al., 1966) and their cell
membranes (Haene-Severns
et
al., 1968); from guinea pig spleen, lung,
kidney, liver (Kahan, 1967; Kahan and Reisfeld, 1967; Kahan et
al.,
1968a) and sarcoma cells (Kahan
et uZ.
19 69 ); from do g spleen cells
(D a ghe r
et
al.,
1967 ); from h um an spleen cells (Ka han
e t
al., 1968b);
from human lymphoid cells grown in long-term continuous culture
(Reisfeld
et
al., 19 70 a); an d from lung, liver, spleen, an d kidney of 3, 4,
and S%-month-old human fetuses (Pellegrino an d K ahan, 1 97 0).
A brief exposure ( 3 5 minu tes) of m urine a nd guinea pig cell sus-
pensions to low-intensity, diaphragm-mediated sound ( 9-10 kc./second;
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TRANSPLANTATION
ANTIGENS 137
15.5
W ./cm.Z; 4"C., Raytheon Model
DF
101 magnetostrictive osciIIator)
liberates 12-15% of the total immunogenic activity of the treated sus-
pensions in soluble form. Titrations of the duration of sonication have
shown that antigen releasc occurs after the cell surface membrane is
disruptcd but prior to complete cytoplasmic and nuclear disruption. This
point correlates with
a
90%decrease in cell count on
a
Coulter counter in
a suspension containing 40-50
x
10" cells/nil. T he dcbris a nd cellular
membranes could be removed by ultracentrifugation at 130,OOOg. It
should be noted that even prolonged centrifugation at 200,OOOg failed
to yield any detectable membrane sediment following ultrastructural
analysis. The antigenic principle from guinea pigs purified by gel filtra-
tion of the supernatc on Sephadex G-200 (0.2
M
tris, 0.5
M
glycine, 0.5%
mannitol, pH 8.0) eluted a t th e front of th e inner volume
( K d
0.92) .
Th e active fraction (Sep had ex Fraction I ) from guinea pigs ( 1 )
induced the specific accelerated rejection of test allografts (K ah an an d
Reisfeld, 1967), (2) elicited specific dclayed-type hypersensitivity re-
actions upon intradermal challenge of allogeneic hosts that had been
presensitized with donor-type grafts (Kahan, 1967), ( 3 ) participated
with sensitized cells in third-party local passive transfer reactions in
syngeneic hosts (Kahan, 1967) or
( 4 )
in irradiated hamsters (Kahan
et
d.
968a) , and (5) stimulated blast transformation of lymphocytes
in vitro (Kahan e t
nl.,
19fBa) (se e belo w).
This Sephadex Fraction I from guinea pig spleen is chemically
complex and contains at least seventeen components which can be re-
solved by discontinuous acrylamide gel electrophoresis at pH 9.4 (Kahan
and Reisfeld, 1967) (Fig. 1) .
Of
these seventeen components, only a
single com ponent ( component 15; R, 0.73-0.74) possesses transplantation
antigenic activity. Component
15
is
imniunogenic-intradermal
adminis-
tration of
1
o 3 pg. of strain 2 component 15 in polyacrylamide gel, which
is known to be a good adjuvant (Raymond
and
W eintraub, 1963 ) , to
allogeneic strain
13
hosts accelerated th e destruction of dono r-type strain
2
grafts but not of strain 13 isografts (K ah an and R eisfeld, 196 9a). T he
antigenic activity of component 15 was
also
demonstrated
by
elicitation
of a specific delayed-type hypersensitivity response by intradermal
challenge of presensitized allogeneic hosts with 0.1 pg. of an tigen (K ah an
an d Reisfeld, 196 7). Th e purified antigen could thus not only ind uce a
state of specific transplantation immunity but also could elicit expressions
of delayed-type hypersensitivity following the induction
of
immunity
by
skin or tumor grafts. Observations such as these strongly suggest that this
component contains a determinant against which hosts develop sensi-
tivity following a llografting.
Com ponen t 15 has been shown to be electrophoretically ho mogeneous
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138
R. A. REISFELD AND B. D. KAHAN
FIG.
1.
Disc electrophoresis pattern of Sephadex Fraction I (pH 9.4, 7.5%
acrylamide gel) applied
350 pg.
of protein.
as
shown by the appearance of
a
single electrophoretic band following
re-electrophoresis of 1251-labeledcomponent 15 ( Fig.
2 )
with and with-
out 8 M urea while varying the porosity of the gel ( 5 , 10, and 15%)
(K ah an an d R eisfeld, 1967,
1%8a,
196913).
The molecular weight for electrophoretically homogeneous strain
2
and strain 13 guinea pig transplantation antigcn was found to be 15,000,
assuming
a
pa rtial specific volume of
0.74. The
three techniques used to
determine molecular weights were (1 ultracentrifugation employing the
Yphantis sedimentation equilibrium method with interference optics, ( 2)
gel filtration in the presence and in the absence of
5
M
guanidine hydro-
chlor ide (Fig. 3) , and 3 ) calculation from the amino acid composition.
The
amino acid compositions of antigens isolated from strain 2 and
strain 13 guinea pigs were quite characteristic and reproducible. Of
interest were the distinct amino acid differences observed between the
electrophoretically homogeneous components 15 prepared from the two
histoincompatible lines of guinea pigs (Table
I ) .
Hexosamine an d half-
cystine were not detectable and methionine was only present in trace
amounts. There were significant differences at the
P
<
0.01
level in the
content of serine, alanine, valine, isoleucine, leucine, and possibly tyro-
sine and phenylalanine. The differences ranged from 1 to 7.5 mole
%
(K ah an and Reisfeld, 1968 b). I n connection with t he recent advances in
the genetic code, it was considered of interest that a single base substitu-
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TRANSPLANTATION ANTIGENS
139
0
0 5 10 15 20
25
45
GEL
CUT N U M B E R
FIG.
2. Re-electrophoresis
of
"'I-labeled strain 2 guinea pig transplantation
antigen (7.5% crylamide gel, pH
9.4).
The gel was cut into forty slices and monitored
fo r radioactivity.
l0,000
5000
0
I
25
I50
200
250
300
EFFLUENT
(rnl.)
FIG.
3 .
Gel filtration pattern of '2?-labeled strain 2 guinea pig transplantation
antigen. The Sephadex G-200 column equilibrated with 0.1 M ammonium bicarbonate
was calihrated with compounds of known molecular weights as indicated.
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140
R.
A. REISFELD AND
B. D. KAHAN
TABLE I
GUINEA
IG TRANSPLATION
NTIGENS
DIFFERENCES
N AMINOACID COMPOSITION BETWEEN 8F RA IN 2
A N I )
STRAIN 13
Difference in
Difference,
No.
of residues,
Amino
St,raiti 2
Stmiii 13 siraiii 2-st~raiil
13
&rain 2-st.rain 13
acid
(mole ) (mole
7c) (mole
5 )
(No./mole)a
Seririe
10.95 18.87
-7.92 -11.1
Alanine
11.40
7.96
$3.44
+4.8
Leu
cine
8 . 5 8
7.19 +7.19 +1.9
Valine
6.46 5.48
+0.98 + 1 . 4
Isoleucine
4.46 3 . 3 2
+1.14
+ 1 . 6
~ ~~
a
Based 011 the
assumption that t here
are
140 residues per mole
of antigen.
tion in serine
(
UCU, UCC, UCA, AGU, AGC) yields isoleucine (AUU,
A U C ) ,
leucine
( U U A , U U G ) ,
an d alanine
( G C U , G C C, G C G )
and tha t
a two-base change yields valine (GUU, GUC, GUA, GUG) which had
the smallest difference considered significant. The remaining amino acids
were present in strikingly similar amounts in the antigens isolated from
the two strains of guinea pigs. These data suggest that guinea pig trans-
plantation antigens possess allotypic specificities related to protein struc-
ture in analogy to the polymorphic genetically segregating antigenic
determinants found on the serum proteins of numerous species. There
seems to be a correlation between the allotypic specificity an d th e amino
acid composition of th e polym orphic substances similar
to
those observed
with rabbit, immunoglobin G, light and heavy polypeptide chains (Reis-
feld et
al.,
1965; Koshland et
al.,
1968). It is reasonable to assume that,
although not all of the observed amino acid differences may be related
to the antigen ic specificity, a t least some of th e am ino acids a re involved
in determ ining th e characteristic immunological properties of these
molecules.
The significance of this protein polymorphism in relation to the
chemical nature of the antigenic determinant becomes even more ap-
parent since there is no detectable lipid or carbohydrate at levels grcater
than 1 . ollowing lipid extraction of component 15 with chloroforni-
methanol (2:1 v/v) and Folch partit ion, thin-layer chromatography was
performed on silica gel G, staining selectively for lipids, glycolipids, and
cholesterol esters (Kolodn y, 196 8a ,b) . Hexose an d pentose content w as
determined b y the cysteine-sulfuric acid metho d ( Dische, 1949).
1 . Hurnnn Spleenic
Antigens
To
preparc solublc HL-A alloantigens, spleens were obtained from
five donors, cell suspensions we re prepared, an d subjected to brief trcat-
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144 R .
A. REISFELD AND
B.
D. KAHAN
1966); 3 ) the observations that extracts from a malignant source were
chemically more complex than those from normal tissue (Kahan
et at . ,
1969); and
( 4 )
he need to have a living donor for long-term continuous
comparisons between his peripheral lymphocytes, his cultured cells, and
the antigen extracted from them. Thus, although the malignant cell lines
offered a temporary solution to the problem
of
a uniform and abundant
source for the extraction of transplantation antigens, the application
of
normal cell lines offers a new dimension to the field.
3 .
Limitations
of
the Sonication Procedure
The use of low-intensity sound to liberate water-soluble alloantigens
has the same major limitations inherent in other solubilization methods.
Thus, the sonication method also solubilizes relatively large amounts
of nonspecific cellular materials together with relatively small quantities
of alloantigens. Sonication does not seem to yield significantly larger
amounts of soluble antigen than do other methods. However, it is
difficult to compare yields reported by various investigators employing
different methods since both procedures and criteria used to evaluate
antigenic activity and specificity differ considerably.
However, the application of low-intensity sound offers some ad-
vantages in that the materials obtained are truly water-soluble as well as
immunogenic and can be obtained as an electrophoretically homogeneous
moiety which is essentially protein in nature. Furthermore, all the
HL-A
alloantigenic determinants detectable on the donor's peripheral lympho-
cytes can be obtained in the highly purified antigen preparation. The
method can, thus, be employed to isolate a chemically well-characterized
homogeneous antigen which can subsequently be cleaved
by
both chem-
ical and enzymatic methods to ascertain the molecular and chemical
nature of the alloantigenic determinants.
E. OTHERMETHODSOR ANTIGENEXTRACTION
Hypertonic salt solutions have been successfully applied to extract
soluble HL-A alloantigens from human lymphocytes in long-term tissue
culture. Cells were washed with Hank's balanced salt solution and then
suspended in Hank's solution containing 3
M
KC1 (10 m1/109 cells)
and stirred gently for 16 hours at 4°C. The extract was then centrifuged
at 130,OOOg for 2 hours and the clear supernate once dialyzed against
Hank's solution contained specific alloantigenic activity as measured by
its capacity to inhibit the cytotoxic action
of
operationally monospecific
alloantisera. The specificity ratios of such crude preparations range from
50
to 100. By this method and with the same salt concentration, from
10-15% of the antigenic activity of the crude cell particulate fraction,
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TRANSPLANTATION
ANTIGENS
145
containing abo ut 1 5 2 0 % f the total cell protein, was solubilized (Re is-
feld and Kahan, 1970).
A cationic detergent, sodium lauroyl sarcostinate, has also bcen used
successfully to solublize HL-A alloantigens from human lymphocytes in
tissue culture. Cells
(
10i/ml.) were homogenized with a Teflon homoge-
nizer (4 °C .) in Hank's balanced salt solution containing 0.7% ( w / v ) de -
tergent. The homogenate was centrifuged at 130,OOOg for 12 hours
( O O C . )
and the supernatant placed at O"C., a temp erature at which much
of the detergent crystallized. The supernatant thoroughly dialized
against Hank's solution specifically inhibited the cytotoxic activity of
operationally monospecific alloantisera. This procedure solubilized from
15
to 20% of the antige nic activity
of
the cell particulate, but contained
only approximately from 3 to 5
of
th e total cell protein (Reisfeld et at.,
1970a,b) .
I l l .
Physical and Chemical Nature
of
Transplantation Antigens
A.
Many investigators have attempted to determine the chemical nature
of histocompatibility antigens. Progress in this was hampered by the
extreme chemical complexity of most antigen preparations studied, and,
consequently, histocompatibility antigens were considered to be almost
every chemical entity known to man. The antigenic determinants were
thus at various times considered to be either deoxyribonucleic acid
(D N A ), lipid, carbohydrate, or protein. Many studies attem pted to
evaluate the effect of a series
of
conditions and reagents on the activity
of highly complex alloantigen preparations. Although such studies were
generally quite useful as a guideline for isolation procedures, the vast
chemical complexity of most of t he an tigen preparations stu die d seriously
limited their value in obtaining conclusive information with regard to
the physicochemical n ature of alloantigenic determinants.
CHEMICAL ATUR E
F
ALLOANTIGENICETERMINANTS
I . Deoxyribonucleic Acid
The pioneering experiments of Billingham et al. (1956b) indicated
that a nuclear subcellular fraction could immunize recipients against
subsequent grafts. Since these authors found that deoxyribonuclease but
not ribonuclease or trypsin inactivated the preparation, they suggested
that DNA determined transplantation specificity. However, neither
Haskova a nd H rubeskova (19 58 ) nor M edawar (1958) were able to
elicit accelerated rejection with purified DNA. Even more conclusively,
Castermans and 0 t h (1956) proved that
a
component other than DNA
carries the ailtigenic specificity when they
found
that rxtraction
of
a
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146 R.
A. REISFELD
A N D B.
D.
KAHAN
nuclear homogenate with sodium chloride yielded an active supernatant
that lacked DNA and an inactive sediment that contained DNA.
2.
Lipid
Intensive efforts by several investigators
(
Herzenberg and Herzen-
berg; 1961; Lejeune
et aE.,
1962; Davies, 1962; Manson et al., 1963) sug-
gested that water-insoluble materials containing approximately equal
proportion of lipid and protein and with a low carbohydrate content
mediated transplantation immunity. These lipoproteins induced acceler-
ated graft rejection, elicited the formation of specific alloantiserums,
and inhibited the reactions of these serums
in
vitro. Davies (1966a)
found that protein precipitated after organic solvent extraction was in-
active and that there was an increasing porportion of lipid with increas-
ing degrees of antigen purification. However, more recently, purified,
soluble niurine alloantigens were found to contain no detectable lipids
or phospholipids (Shimada and Nathenson, 1969). It has become quite
apparent that more purified materials containing little or no detectable
lipid possess all
of
the attributes of transplantation antigen (Kahan and
Reisfeld, 1967, 1969a, 1969b; Kahan et al., 1968b) and that lipoidal
fractions by chloroform-methanol or ethanol extractions have no allo-
antigenic activity (Graff and Kandutsch, 1966).
3.
Carbohydrate
Billingham
et
al. (1958) showed in later work antigenic activity in
the sediment of cells that had been exposed to ultrasound and centrifuged
at
27,OOOg.
The antigenic determinant in this preparation was proposed
to be a mucoid since its biological activity was drastically reduced by
two reagents: (1 receptor-destroying enzyme, a complex mixture from
Trichomonus foetus,
and ( 2 ) periodate (0.005-0.01
M ) .
Several other
investigators ( Kandutsch and Reinert-Wenk, 1957; Kandutsch and Stimp-
fling, 1966) felt that carbohydrate moieties could be part of the antigenic
determinants of transplantation antigens since they found inactivation
following periodate treatment. However, none of these studies clearly
demonstrated specific effects on the carbohydrate moiety per se, e.g.,
specific oxidation products, and at the same time they documented that
periodate did not adversely affect amino acids and, thus, protein con-
figuration. Kandutsch and Stimpfling (1966) have shown that when
mouse transplantation antigens are exposed to periodate (0.001M ) , there
are marked changes in the content of several amino acids including tyro-
sine, isoleucine, and leucine. It is, indeed, difficult
to
ascribe the nature
of the antigenic determinant to carbohydrate on the basis of the reaction
of highly complex mixtures with relatively nonspecific oxidizing agents
such as periodate.
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TRANSPLANTATION ANTIGENS
147
In further attempts to implicate carbohydrate as the antigenic de-
terminant of histocompatibility antigens, Brent et
al.
(1961)
found tha t
some polysaccharides with Forssman affinities-blood gro up su bsta nc e A
(b ut not B, H, or Le a) , Type XIV pneumococcal polysaccharide ( b u t not
Types I, 11, or
V ) ,
and Shigella shigae
polysaccharide-inhibited
t he
agglutination of erythrocytes by alloantiserum in a fashion analogous to
the hapten inhibition observed with blood group isoantiserums. Davies
( 1966 a) fou nd t ha t D-galactopyranose-p- (
1
+ 4 ) D-glucosaminoyl residues
partially inhibited alloantiserum against specificity H-2 18
( R )
and tha t
N-glycolyl neuraminic acid specifically inhibited some mouse alloanti-
serums. However, these studies have not been continued and have at
least thus far failed to yield much insight into the chemical nature of
the determinants, since the observed effects were generally very weak.
Shimada and Nathenson
(
1969) found that papain-solubilized allo-
antigens from spleen cells of m ice with H -2” an d H-2d genotype are
are glycoprotein with an 80-90% protein moiety. They base their claim
on total neutral carbohydrate estimations by the orcinol method and by
determinations of sialic acid and glucosamine. These determinations
were related to dry weight. However, the amount of purified material
was so small that dry weight could not be determined, and a 90%value
for the protein content of the dry weight had to be assumed. On the
basis of the se assumptions, ne utra l carbohydrate was calculated to range
from 3 to
5 ,
hexosamine from 3 to 4.4%)and sialic acid from 0.9 to 1.3%.
Whether or not these carbohydrate moieties are part of the antigenic
determinant per
se
has not been determined thus far.
D.
L. Mann et
al.
(1969b) used papain to solubilize human allo-
antigens from two cell lines bearing different alloantigenic specificities.
These alloantigens were found to contain from 5 to 8%orcinol-reactive
carbohydrate.
N o
hexosamine could be detected, which is curious since,
with the exception of collagen, there are no known mammalian glyco-
proteins that lack hexosamine. In fact, recent studies have shown that
hexosamine is present in human platelet membrane glycoproteins
(Pep-
per and Jamison, 1969). As pointed out by D. L. Mann
et u Z .
(1969b) ,
there is as yet no decisive information, which can be drawn from their
data, indicating that the carbohydrate portion is responsible for allo-
antigenic specificity of HL-A antigens.
4. Protein
There is a considerable body of evidence which suggests that poly-
peptide is essential for antigenic activity. Thus, Kandutsch and Reinert-
W enk 1957) observed irreversible destruction of antigenic activity afte r
exposure to protein denaturants, e.g., 50%urea, 90 %phenol, aqueous
alcohol, heat, and pH values less than 4 and greater than 9. Kandutsch
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TRANSPLANTATION ANTIGENS
149
other than proteins make
up
the vast majority of antigenic determinants
of human and mouse alloantigens. One
can,
of course, argue that ( 1 )
polypeptide configuration is crucial to express alloantigenic determinants
composed of carbohydrates or
( 2 )
carbohydrates are instrumental in
bringing about a given polypeptide configuration which expresses a
certain alloantigenic specificity. Neither of these arguments seems par-
ticularly compelling in view
of
the observation that electrophoretically
homogeneous guinea pig transplantation antigens and human alloantigens
do not contain either carbohydrate or lipid at the
1%
imit of the analytical
method (Kahan and Reisfeld, 1968b, 1969b). Thus, in the case of guinea
pig antigen (molecular weight
15,000)
there is at most one residue, and,
in the case of human antigen (molecular weight
34,600),
there are at
most two residues of carbohydrate per molecule. These hypothetical
values are advanced only because it is simply impossible with available
analytical methods to rule out absolutely the presence of a very small
carbohydrate moiety in a relatively large molecular weight protein
molecule. On the other hand, amino acid analyses of electrophoretically
homogeneous transplantation antigens from two histoincompatible in-
bred strains of guinea pigs strongly suggest that their antigenic de-
terminants depend upon protein structure because they show marked
and reproducible differences in their amino acid composition (Kahan
and Reisfeld, 1968b). Although it is not presently known whether
the
antigenic products of the two histoincompatible, inbred strains examined
are the result of single or multiple gene differences, the data suggest
that these transplantation antigens possess genetically segregating (allo-
typic) specificities related to protein structure possibly in analogy to
the polymorphic antigenic determinants found on the serum proteins
of numerous species. Finally, the mediation of transplant rejection by
a cell-bound immune response implicates polypeptide specificities since,
as Holborow and Loewi (1967) have summarized, “there is practically
no evidence that man or animals develop delayed type hypersensitivity
toward polysaccharides, and in that respect they differ sharply from
proteins.”
Furthermore, the distinct and reproducible amino acid differences
observed between transplantation antigens prepared from two histo-
incompatible lines of guinea pigs suggest that these antigens possess
allotypic specificities related to protein structure ( Kahan and Reisfeld,
1968b). The polymorphism of the primary protein structure of these
antigens
is
strikingly similar to that observed in rabbit immunoglobulin
light chains with different allotypic specificities (Reisfeld et al., 1965).
Since histocompatibility antigens are readily distinguishable products of
well-defined alleles which are distributed among some but not all mem-
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150
R . A.
REISFELD AND B. D. KAHAN
bers of the same species, these polymorphic forms of genetically segregat-
ing antigenic determinants can be considered to form an allotypic system
analogous to that described for serum immunoglobulins
(
Oudin, 1966).
I n both cases ther e is a correlation betw een allotypic specificity an d
the amino acid composition of these polymorphic substances.
It is impossible at this point to rule out that either lipid or carbo-
hydrate moieties express a specific alloantigenic specificity
per
se or
that they confer a unique protein configuration that determines such
a specificity. There is little doubt that carbohydrate moieties present
on cell membranes and on some alloantigens may have an important
physiological role which will probably become apparent from future
research efforts. Ho we ver, a t prese nt, it seems th at most, if not all,
antigenic determinants of alloantigens are polypeptide in nature in view
of the data summarized here and since the large number of amino
acid sequence analyses and immunogenetic studies of human, mouse,
an d rabb it immunoglobulins thus far show tha t only polypeptides express
genetically segregating antigenic ( allotypic) determinants.
B. PHYSICAL ATU RE F ALLOANTICENS
1 .
Molecular Heterogeneity
of
Alloantigens
The notion that alloantigenic determinants might
be
associated with
different molecular species of the cell membrane received some of its
stimulus from the proposal that two closely linked chromosomal regions
may determine th e genetic control of H L-A alloantigens (C ep pe llini
et al.,
1968; Kissmeyer-Nielsen
et
al., 1968; Dausset
et
al., 1969) and from the
work of Boyse et al. (19 68 ) indicating tha t H-2d an d H-2k alloantigens
were distant from each other on the membrane of mouse thymocytes.
Several investigators observe d tha t following solubilization by papain,
both human and murine alloantigens could be fragmented into entities
that lacked some of the specificities of the donor and, in some cases,
contained only a single detectable antigenic specificity as determined
by
their ability to inhibit the cytotoxicity of a very limited number of
operationally monspecific alloantisera ( Sanderson, 1968; Davies, 1969;
D . L. Maim
et
al., 1969a,b) .
Davies
(
1969) used either autolytically or papain-solubilized antigen
extracts to show tha t by chromatography on D EAE-Sephadex columns
he could differentiate, from very complex protein effluents patterns,
various molecular species carrying only one or, in some instances, several
H-2 specificities. Some H-2 antigens of a single phenotype are obtained
in this manner. Thus, from H-2” mouse antigen, H-2.5 specificity was
obtained essentially free from H-2.8 and, in turn, H-2.8 was obtained
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TRANSPLANTATION ANTIGENS 151
f ree f rom H-2.5. These antigenic spccificitics were separated by DEAE-
Sephadex chromatography, applying a simple straight-line salt gradient.
Sanderson
( 1968)
initially failed to separate
HL-A
alloantigenic speci-
ficities by ge l filtration
on
Sephadex G-200. Instead, alloantigenic specific-
ities which had a relatively high specificity ratio (SR + 100) emerged
together in a region where calibration curves indicate
a
molecular weight
of 45,000. A smaller peak of aggregated material was found in the ex-
cluded volume containing thc same antigenic specificities but with
a very low specificity ratio ( S R +
1 ) .
This observation is of some in-
terest in view of the data of D. L. Mann
et uI.
(1968, 1969a,b) who al-
ways found antigenic activity (i n some cases the m ajority) in th e ex-
cluded volume of
a
Sephadex
G-150
column; however, these investigators
have not reported any specificity ratios for their separated alloantigens.
D.
L.
Mann et
al. (1969a)
claimed molecular heterogeneity of human
alloantigens based on their ability to separate alloantigenic determinants
from both subloci HL-AQ, HL-A4, and HL-A7, respectively. It is inter-
esting that all alloantigenic specificities analyzed were found both in
the excluded an d included volumes. However, mo st of t he HL-A2 activity
was in the excluded volume whereas most of the HL-A4 and HL-A7
activities were found in the included volume of the Sephadex
G-150
eluate. Moreover, although somewhat overlapping, the peak values
of
HL-A4
and
HL-A7
activities appeared in slightly different elution posi-
tions
( n o elution volumes or K d values were given) with a smaller
peak of HL-A4 activity ap pe arin g in yet ano ther elution position to-
gether with small amounts of
HL-A2
a nd
HL-A7
activities.
In a more recent study of the sam e lymphoid cell line
(R-4265) ,
D. L.
Mann et al. (1969b) showed a Sephadex G-150 elution profile which de-
picts only one alloantigenic specificity
(HL-A2)
determined by an opera-
tionally monospecific antiserum . Th e majority of this specificity was again
found in
the
excluded volume. The area of the effluent pattern in which
previously
( D .
L. Mann
e t
al.,
1969a)
several alloantigenic specificities
were shown to be resolved was now covered by a large peak indicating
specificities by a highly polyspecific antiserum ( Iochum ) which reacts
with lymphocytes from
96%
of a normal population. Within this activity
region of the included volume, also a small peak
of
HL-A2 specificity
appeared, It is surprising that apparently separated components with
different alloantigenic specificities could not be separated at all on
acrylamide gel electrophoresis but appeared within one electrophoretic
zone. Shimada and Nathenson (1967) found that, by Sephadex gel filtra-
tion, murine alloantigens could be separated into entities with different
H-2
specificities. In a more recent study (Shimada and Nathenson,
1969),
it
was observed that, although this was not possible for H-2'
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152
R . A. REISFELD
AND
B. D.KAHAN
alloantigens, there was some separation of H-2') alloantigenic specifici-
ties H-2.5 and H-2.2, both of which were in the included volume of a
Sephadex G-150 column. However,
it
was
not possible to separate these
alloantigenic specificities by acrylamide gel electrophoresis. Both of the
purified preparations
(
H-2'' and H-2d) resolved into three electrophoretic
components each
of
which carried essentially the same combination of
antigenic specificities. In order to reconcile these electrophoretic analyses
with the gel filtration data, one has to propose that electrophoresis re-
solved components with different overall charge properties from each
of the two antigen preparations. It is probable that, since only a 7.5%
acrylamide gel was used, most separation occurred on the basis of charge
rather than size. Since the electrophoretic components have basically
the same antigenic profile and also relatively similar amino acid com-
positions, it seems, indeed, feasible that they represent a series of frag-
ments produced by papain. It is possible that antigen separation on
Sephadex occurs due to the association of antigen fragments with other
proteins and that these protein-protein interactions are minimized by
the conditions
of
acrylamide gel electrophoresis.
From the proposal that at least two closely linked chromosomal re-
gions determine the genetic control of
HL-A
alloantigens (Ceppellini
et
al.,
1968; Kissmeyer-Nielsen
et
al.,
1968; Dausset
et
al.,
1%9), one can
readily deduce that either
1 )
wo structural cistrons code for separate
molecules with either one or the other series of determinants or (2) one
structural cistron with multiple mutational sites is able to
do
the same.
Although such hypotheses (D. L. Mann e t
al.,
1969a) may eventually
be useful to understand the genetic mechanism that controls the ex-
pression of histocompatibility antigens, it seems that the lack of chemi-
cally well-defined alloantigen molecules makes it difficult at present to
test such hypotheses at the molecular level.
Several hypotheses can be advanced in an effort to explain the finding
of alloantigenic specificities on components separable by either gel filtra-
tion (D. L. Mann et al., 1968, 1969a,b; Shimada and Nathenson, 1967,
1969)
or
by cellulose or Sephadex ion-exchange chromatography ( Sander-
son, 1968; Davies, 1969). Since a nonspecific proteolytic enzyme such
as papain is allowed to react in large excess with a highly complex mix-
ture of cellular proteins, it is conceivable that the enzyme cleaves in
a random fashion, not only materials containing antigenic determinants
but also other proteins, Protein-protein interactions and aggregation
could account for some of the separations observed on gel filtration and
ion-exchange Chromatography. Fragments containing alloantigenic de-
terminants could certainly associate with proteins bearing no alloantigen
determinants which may or may not have been cleaved by papain. When
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TRANSPLANTATION ANTIGENS 153
proteins are cleaved into different fragments, their binding capacity for
other substanccs also may change, furth er complicating the picture. T he re
is little doubt that, following proteolysis, ccll membrane fractions,
whether separated by gel filtration or ion-exchange chromatography, are
highly complex mixtures (Halle-Panenko et
ul.,
1968; D. L. Mann et ul.,
196913; Shim ada an d Nathenson, 19 69 ), an d it is no surprise that the
same antigenic determinants often appear in different portions of a
chromatogram. Furthermore, since papain can apparently easily destroy
alloantigenic activity (Sanderson, 1968; D. L. Mann
et
al . , 1969b;
Shim ada an d Nathenson, 19 69 ), it seems qu ite feasible tha t this non-
specific proteolytic enzyme may produce a number of fragments that
lack certain antigenic specificities, and that then can be resolved by
either gel filtration or ion-exchange chromatography. In fact, it does not
seem unreasonable to assume that papain, which can easily destroy
membrane-bound specificities ( Shimada an d N athenson, 196 9), can also
“silence,” i.e., selectively inactive, certain alloantigenic determinants
m ade more susceptible once
a
given antigenic fragment is removed from
the membrane and is then exposed to the large excess of papain present
during the 1-hour digestion period.
Although the molecular heterogeneity of alloantigens certainly poses
a
challenging and intriguing problem, it is difficult to draw any mean-
ingful conclusions on the basis of presently available experimental evi-
dence. It would probably be profitable to solubilize alloantigens without
the aid of nonspecific proteolytic enzymes and then apply chemical and
enzymatic methods only to thoroughly purified and characterized entities
in an attempt to separate and characterize fragments bearing different
alloantigenic determinants.
2 .
The Homogeneity Problem
The selection of criteria for homogeneity of polymorphic proteins
and glycoproteins solubilized from cell membranes poses some prob-
lems. As pointed out previously, all the methods presently in use suffer
from a lack of selectivity, i.e., they tend to solubilize large amounts of
complex materials from the cell membrane containillg relatively small
quantities of alloantigens. This leaves one with the “needle in the hay-
stack” problem, the end result of which is in the best case, the isolation
of an extremely small qu an tity of antigenic m aterial. T his places
a
severe
limitation on rigorous determinations to establish homogeneity of this
material. Consequently, the only techniques used are those that require
little material, i s . , acrylamide gel electrophoresis a nd analytical ultra-
centrifugation.
Discontinuous acrylamide gel electrophoresis is, when properly exe.
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154
R.
A.
REISFELD
AND
B.
D.KAHAN
cuted,
a
sensitive technique with excellent resolving power. The method
is most discriminating and is at its best when used with varying acryl-
amide pore sizes, urea, and radiolabeled samples; it
is
a
poor criterion
of electrophoretic homogeneity when these parameters are not applied.
Th en “bandsmanship” takes over, i.e., any “single b a n d is interpreted
as
a sign of homogeneity, no matter whether it is barely visible or whether
there is an und erlying or tailing sme ar of stained bu t unresolved proteins.
Shimada and Nathenson (1969) found that murine alloantigen prep-
arations, which ha d b een purified b y gel electrophoresis an d ion-exchange
chromatography, when applied to acrylamide electrophoresis showed
three components
( R f
0.36, 0.38, and 0.40). These components were
poorly resolved and contained much underlying and tailing material.
Re-electrophoresis of one of these
H-2”
components ( R , 0.38) in the
presence of sodium dodecyl sulfate
(SDS)
resulted in the appearance
of a single 2-3-mm. wide zone which in itself was co nsidered an indica-
tion of electrophoretic homogeneity. It was pointed out that
H-2d
elec-
trophoretic components showed, und er th e same condition, an additional
component, estimated as
10%of
the total, based solely on its dye in-
tensity. Estimate of so-called “minor components” based simply on dye
intensity have often proven to
be
erroneous especially when the band
ap pe ar s diffuse. Estim ates of componen ts based on scann ing of gels
containing radiolabeled materials has proven to be
a
much more reliable
method. The choice of SDS gel to indicate electrophoretic homogeneity
is rather unfortunate since the strong negative charges present on the
detergent overwhelm all charge differences,
so
that any small charge
differences between protein components are hidden and only large size
differences can resolve proteins in this system. Incidentally, this electro-
phoresis system also lacks the discontinuous voltage gradient which is
the key to th e s ha rp resolution
of
disc electrophoresis. Furthermore, SDS
gels, to be most informative, should be run at a number of different
acrylamide concentrations to make full use of the size resolving power
which is th e main att rib ut e of these gels. As shown previously (Kahan
an d R eisfeld, 1 96 9b ), to m ake th e m ost efficient use
of
the high resolving
power of the discontinuous acrylamide system, it is useful to radiolabel
the material to be characterized and then to carry out electrophoresis
in urea, followed by the screening of narrow gel slices. Only a sharp,
single peak resolving within o ne gel slice is indicative of electrop hore tic
homogeneity. Moreover, since the radiolabel has
a
sensitivity of several
magnitudes greater than the Coumassie dye, one can evaluate much
more accurately the existence and quantitative extent of an underlying
or tailing unresolved protein zone. These criteria previously applied by
others (Ka han a nd Reisfeld, 19 69 b) to test th e degree of electrophoretic
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TRANSPLANTATION ANTIGENS 157
These observations, together with those indicating that only a minor
portion ( 1 5 2 5 % ) of the alloantigens can be solubilized by existing
methods, make it necessary to postulate that there are some antigens
that can be solubilized relatively easily, whereas others resist solubiliza-
tion by presently available methods. In the light of the foregoing dis-
cussion concerning the nature of the cell membrane, it seems feasible
that antigens associated with macromolecular entities on the membrane
by means of ionic bonds or hydrogen bonds may
be
solubilized by either
proteolysis, detergents, sound, or hypertonic salt extraction.
On
the other
ha nd , man y antigens cannot be solubilized by any of these treatments an d
could, thus, be located within the apolar core of the membrane, highly
interacted with hydrophobic lipid moieties. It is obvious that at present
no real conclusions can
be
drawn and that considerable research will
have to be done to understand the physicochemical nature of the at-
tachment of histocompatibility antigens to lymphocyte cell membranes.
IV.
Biological Activity of Extracted Transplantation Antigens
The ability of tissue extracts to accelerate the destruction of donor-
specific grafts
(
Billingham et al . , 1956a,b), the second-set phenomenon
( Medawar, 1944), demonstrates the activity of these extracts as trans-
plantation antigens. Although this system most meaningfully reflects the
role of the substances in histocompatibility, it is
a
cumbersome, insensi-
tive, only crudely quantitative, and time-consuming technique for the
screening of putative antigens, Therefore, more rapid, flexible systems
dep end ent upon delayed-type hypersensitivity or upon humoral antibody
have been routinely employed for antigenic detection.
A.
TRANSPLANTATION
OMPATIBILITYSSAYS
I,
Accelerated Rejection
of
Allografts-Skin Transplants
Loeb ( 1930) suggested that if transplantation resistance depended
upon
an immune mechanism, a second graft transplanted onto
a
host
when his reaction against the first graft from that donor was at its
height, would
be
attacked with equal intensity at once. Medawar (1944)
succeeded in demonstrating that challenge grafts obtained from the
donors of the sensitizing transplant were destroyed more rapidly than
transplants from third-party donors and that the resistance induced by
allografting was systemic. Billingham
et al. (1956a)
showed that pre-
treatment with subcellular extracts immunized recipients specifically
against donor-strain skin grafts and, thus, established that transplanta-
tion
antigenic activity did not depend upon intact cells.
In routine practice, an antigenic preparation is administered to an
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158
R. A, REISFELD A N D B. D. KAHAN
I80
160
80
60
20
“I
d & L Z s T 0 f
.
OT (0)CRE
7,8
I 1 ~ 1 2
15j6
9,IO
13J4
17,18
S L I C E N U M B E R
FIG.6.
Cutaneous reactions of preimmunized individuals
SGH,
BIA, CAP and
of normal individuals
BOT
and ROC, to acrylamide gel cuts after electrophoresis of
NAV Sephadex Fraction I.
allogeneic host and, after an interval of a few days, the recipient is
challenged with grafts derived from the donor strain and from a third-
party strain, i.e., a strain different than that of the donor or the recipient.
In Fig. 6, the host has received soluble antigen extracted from the
spleens of the strain of the left (upper) graft and later was challenged
with skin transplants from the donor strain and from
a
third-party
strain (the right lower graft). The specific accelerated rejection of the
upper graft in this figure revealed the presence of transplantation anti-
genic substances in the original extract, There are several significant
factors in the performance
of
this assay.
a. Route of Administration of Antigen. Medawar (1957) demon-
strated that in rabbits administration of leukocytes
by
the intravenous
route was less efficacious than b y the intradermal route in the induc-
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TRANSPLANTATION ANTIGENS
159
tion of allograft immunity. In th e late r work of Billingham an d Spa r-
row, intravenous injection of trypsin-dissociated epidermal cells resulted
in prolonged skin graft survival, whereas intradermal administration
of these cells resulted in accelerated rejection of the transplants. The
phenomenon was confirmed in the case of pretreatment of rabbits
with spleen cells
(
Billingham
et al . ,
1957). Inde ed, Merrill and colleagues
(1961) failed to detect sensitization of human beings preimmunized
with intravenous leukocyte injections.
This
difference is not as pro-
nounced in the mouse (Billingham
et
aZ.,
1957) : administration of rela-
tively high doses of spleen cells (250,000 a nd 5
x
l o6 cells) were equally
effective by t he intravenous subcu taneous, intramu scular, intradermal,
or intraperitoneal routes. By the intraperitoneal route
2000
cells provoked
just perceptible immunity, and in the range of 2000 to 5
x lo6
cells the
dose-response curve ap pe are d quite flat.
In
the guinea pig the intra-
venous and intraperitoneal routes were equivalent at the
50
x l o 6 cell
dose (Billingham
e t
al., 1957).
A. P. Monaco
et
al. (1965) found no significant difference
in
ei ther
the degree of allograft immunity induced or in the time course
of
its
development following intravenous
or
intraperitoneal administration of
a membranous transplantation antigenic fraction prepared by homogen-
ization. In contradistinction to these particulate materials, the result
obtained after administration of “semisoluble” or soluble antigenic frac-
tions depended upon the route of administration. The weight of evidence
with transplantation antigens, just
as
with a variety of other immuno-
logically potent materials, indicates that solubilized materials tend to be
tolerogenic when administered by the intravenous route and immuno-
genic when administered by the subcutaneous route. The superiority
of
the subcutaneous route for the administration of transplantation antigens
was established
in
murine systems with sonicated (Kahan,
1965)
and
detergent-solubilized materials ( Graff and Kandutsch, 1966). Further-
more, Brent
et
al. (1962b) demonstrated that severaI soluble prepara-
tions di d no t sensitize when adm inistered intravenously-a situation th at
parallels th e depen den ce of th e Sulzberger-Chase phenom enon on th e
portal vascular system. Further studies on tolergenicity of antigens (as
discussed below) have employed the intravenous route. Immunogenicity
studies, on the other hand, generally have utilized the subcutaneous or
intraperitoneal routes of administration.
Len gth of Interval b etw ee n Antigen Adnzinistrution
and
Chal-
lenge
Skin
Grafting.
T h e kinetics of t he induc tion of hom ograft imm unity
are poorly understood. There is little doubt that the sensitizing activity
and the permanence of the immunity
so
induced depend upon the form
of
the transplantation antigen employed for immunization.
Thus,
sensi-
b .
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TRANSPLANTATION ANTIGENS
161
the challenge grafts. When cells were administered on the day of, or
1
day after, graftin g there was accelerated rejection w ith less but
still
a
perceptible e ffect when administered on th e third d ay a fter
grafting.
Administration of subcellular transplantation antigen produce a more
attenuated immunity. Although A.
P.
Monaco et al.
(1965)
found tha t
the sensitizing capacity of a
105,000-G.
sediment of homogenized cells
was just as gre at as tha t of an equivalent nu m be r of cells, most investiga-
tors found the extracted antigens to be much less potent than intact
cells (Brent,
1958;
Celada and Makinodan,
1961;
Kahan,
1965).
Quan-
titative studies on murine sonicated antigen revealed that although
280,000 whole spleen cells were required to induce immunity against
allografts,
10.7
x
lo6
sonicated cell equivalents, about thirty-five-fold
greater, were required to induce the same degree of sensitization (Kahan,
19Ma) .
The difference in potency between whole and disrupted cells
has been at tr ibuted to
( 1 )
the destruction of labile antigens or their
carriers (Brent,
1958),
(2 ) the increase in antigen dosage due to cell
division of viable cells, 3 ) a long er persistence of viable cells in th e
host resulting in a more prolonged antigenic stimulus (Celada and
Makinodan,
1961),
( 4 ) the migratory capacities and homing tendencies
of intact lymphocytes ( Fichtelius,
1958;
Murray,
1964;
J . Bainbridge
et
al.,
1966), and
(5)
th e possible greater potency of transplantation
antigen located on intact cell surfaces to interact with cell-bound
recognition systems.
Indeed, there is
a
further decline in immunogenic activity as the
antigenic principle is purified from the disrupted cell fractions. Partially
purified antigenic fractions show not only a rapid induction of immunity,
similar to that seen with the administration of dissociated cells, but
also an ab ru pt d ecline in sensitivity. Th e ra te of d ecline in sensitivity is
inversely proportional to the dose of antigen administered, but in all
cases reported, there is less than
10%
of the original activity at
14
days
after antigen administration ( Kahan,
1964a,b).
Furthermore, of the im-
munogenic activity of the total nonviable cell,
22%of
the activity can be
detected in the
105,OOOg
sup erna te of sonicated cells ( in a soluble form )
i.e.,
48
x lo6 cell equivalents of soluble antigen are required to equal
the immunogenic
effect
of
10.7 x
10'; sonicated cell equivalents (Kahan,
1964a). Although it is possible that in the course of purification a sub-
stantial proportion of the activity is lost due to denaturation of the anti-
genic material, it is also possible that contaminant materials without
immunological specificity appreciably contribute to the imlnunogenic
effect of the active principle. In this regard, Messina and Rosenberg
(
1962)
found the delayed-type hypersensitivity response to egg albumen
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TRANSPLANTATION ANTIGENS
163
Although some investigators have relied upon gross observation of
graft survival (Billingham et
a/.,
1956a; Manson et
al.,
1963;
A. P.
Monaco
et
al.,
1965)
as
an ap pro pr iate mea sure of immunity, histological
scoring is generally felt to be a preferable method (Billingham e t al.,
1958; Brent et al., 1962a; Kahan, 1965; Al-Askari et al., 1966). The sur-
vival en d point of skin allografts can only be determined with acceptable
accuracy by naked-eye inspection if th e laten t period before tissue break-
down lasts at least 10 days, so that the graft can cast off its cuticle and
original pelt of hair and expose its epithelial surface to direct inspection.
In the case of rapid and violent rejections across strong histocompatibil-
ity differences, the prerequisites of naked-eye inspection are not fulfilled,
an d histological examination of t he epithe lial survival is essential ( Billing-
ham and Medawar , 1951).
Several methods
of
histologically assessing and classifying graft
survival have been described. Billingham et al. (1954a) provided an
accurate method to compute survival times based upon histological
analysis of serial sections of the challenge graft to determine the overall
epithelial survival. By application and statistical methods, they obtained
a mean survival t ime (M S T ) which was analogous to the LD,, method
of drug toxicity studies.
The histopathology of the allograft reaction is not well understood.
Histological examination of first-set grafts reveals that the immunological
attack against them begins asynchronously throughout the transplant.
Inflammatory changes and infiltration of host leukocytes are maximal
a t about 6 days. At 1 to
3
days later, the re is the beg inning of o vert tissue
breakdown and, after an additional 3-4 days, the process is completed,
showing dilatory progress of the phenomenon after the reaction has bee n
initiated (Billingham et
al., 1954b).
A
point of interest and a source of considerable confusion was the
observation that the histopathology of second-set reactions could differ
quite remarkably. Billingham et al. (1954b) showed that the homo-
graft on a normal, nonimmune mouse had an inflammatory and infiltra-
tive reaction, i.e., an internal phenomenon, whereas the homograft reac-
tion on an immunized mouse occurred prior to revascularizatioll at the
graft-host interface. Animals who ha d not been rendered imm une by
administrative of
a
putative antigen, underwent transient proliferation
of the graft epithelial layer without evidence of cell death at the sixth
postgraft day. In contradistinction, other investigators described the
histological pattern of second-set reactions as an acceleration of the
processes of firstset rejection, viz., infiltration of the graft with lympho-
cytes and temporary hyperplasia of th e graft (E ich w ald an d Lustgraaf,
1961; Andre et d. 1962).
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164 R.
A.
REISFELD AND
B.
D.
KAHAN
Indeed, these two conditions were demonstrated by Eichwald et al.
(1966) to be polar cxtremes of a single continuum. They found that tail
skin grafts applied 10 days following sensitization with either skin
grafts or with
20
X l o Gspleen cells showed one of three typical histo-
logical appearances: I white graft-a particularly intense reaction in
which there is no vascularization of the skin graft; ( 2 ) red graft (hemor-
rhagic necrotic) revealing engorgement, hemorrhage, epithelial necrosis,
and lack of dermal infiltration; and (3) blue (
infiltrative-hyperplastic
grafts without much change in the epithelium but with a premature
subdermal activity when examined at 6 days after grafting. Stetson
(1959) initially felt that white graft immunity differed qualitatively from
accelerated rejection responses in that the former was due to circulating
antibody which could be transferred with serum, whereas the latter
was an anamnestic response mediated by cell-bound antibody. However,
some investigators (1 have failed to transfer white graft immunity with
serum (Brent
e t al.,
1959; Eichwald
et
al., 1966),
( 2 )
have been able
to transfer white graft immunity with cells (Eichwald
et
al., 1966), and
( 3 ) have noted the incidence of white grafts to be high even in donor-
host combinations in which antibodies have not been demonstrated after
grafting. Eichwald
et
al. (1966) demonstrated that from a genetic view-
point, red and white grafts did not differ but reflected quantitative differ-
ences. Not only could
H-2,
but also non-H-2 antigenic differences yield
white grafts, e.g., 518
DBA-2
grafts on Balb-c mice were white, and
data on
F2
hybrids suggested that this effect was due to the cumulative
action of multiple weak loci. Thus the incidence of white grafts depends
upon the number and type of antigenic differences between donors and
recipients. Blue grafts were seen in congenic lines differing at the H-1
and H-4 loci. Interestingly, the result across the Y male chromosomal
antigen varied with the technique of sensitization: intraperitoneal injec-
tions
of
splenic cells produced blue grafts; subcutaneous injections with
challenge at 8 to 15 days produced red grafts; and subcutaneous sensi-
tization with longer intervals before challenge yielded blue grafts.
In
addition to the histological method of assessment of
graft
survival,
there are two other acceptable techniques. In the stereoscopic method the
blood flow through the subdermal vessels is assessed in order to de-
termine the time of circulatory standstill. This survival time is approxi-
mately 2 days less than the histological mean survival time (Taylor and
Lehrfield, 1953a,b). The third method of assessment, also of an all-or-
none character, is the survival test
of
Medawar (1944). The challenge
graft which has resided on the sensitized animal is transferred back
onto a member of the donor strain and observed for its survival in an
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TRANSPLANTATION ANTIGENS 165
isogenic environment. A graft that has been completely destroyed can
thus be distinguished from :i graft with some surviving epithelium.
It is of no small consequencc that appreciable variations occur in
the responses of the recipient animals. Obviously, the strongcr the anti-
genic stimulus, th e less th e deg re e of variation. Billingham
et al.
(1954b) assessed some of the factors affecting variability in the second-
set assay system. Although residual heterozygosity within inbred lines
is possible, it has been found to be minimal in strictly inbred lines
evaluated at a single point in time. However, i t is known that there
can be differences between sublines and even secular drift in the anti-
genic constitution of a single subline examined over 10 to
20
generations
(Gr une berg , 1954; Hildemann
e t
al.,
19 59 ). It appears tha t differences in
graft dosage are minor effects (Me da w ar, 19 45 ), except when specifically
examined over a n exceedingly wide rang e of graft sizes. Th er e are differ-
ences in the physiological state of the graft, e.g., different stages in the
hair activity cycle, an d in th e physiological state of the ind ividual, e.g.,
as related to stress status since corticosteroids are known to affect sur-
vival (Sparrow , 1 95 4) . However, th e primary source of variation is in
the surgical technique affecting healing and vascular penetration of
grafts.
d .
Dose-Response Relationships. Billingham et
al. (
1957) found
a
rough correlation between the number of spleen cells administered
and the severity of the second-set reaction. L. T. Mann e t al. (1959)
and Corson et al. (1967 ) found
a
direct relation between the histological
extent of destruction, employing six distinguishable classes of epithelial
survival, and the logarithm of the number of cells administered. They
appeared to be able to measure a differential response over a twenty-
fold range from 50,000 to
1x
l o o cells. These findings were confirmed in
the rat by Steinmuller and W einer (19 63 ). On the other hand, employ-
ing gross inspection to determine the extent of epithelial survival, A. P.
Monaco et
al. (1965)
found that large differences in the dosage of
membranous antigenic fraction reflected only small differences in graft
survival. Quantitative studies on sonicated murine antigens revealed
linear relationships between histological classes and the logarithm of the
cell dosage, facilitating assessment of the extent of purification, the
kinetics of sensitization, and the reproducibility of the response to s o h -
bilized antigens
(
Kahan,
1965).
In the low dose range, the immunological response to sensitization
appears to be graded in linear fashion. The administration of allogenic
cells in divided doses rather than in single doses produces
a distinct
dose-response relation. Hildemann et al. ( 1959) administered 300 whole
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TRANSPLANTATION ANTIGENS 167
to its polymer state could b e used as a powerful immunogen, and antigen
prepared in the monomer state would fiinction as an effective tolerogen.
2.
Accelerated Rejection
of
Allografts-Lymphoid
Tissue Transplants
Deu tsch (1 89 9) showed th at splenic grafts from guinea pigs actively
immunized against
Bacillus typhosus
caused the transient appearance of
agglutinins in low titer in the normal hosts into which they had been
implanted. I n the murine system, Mitchison (19 57 ) was ab le to dis-
tinguish individuals preimmunized against donor-strain tissue from
those individuals who were nonimmune by the rapid decline in adop-
tively acquired antibody production in the former group. This assay was
used by Celada and Makinodan (1961) who transferred 24
x
l o G pleen
cells from donor mice primed with sheep red blood cells into heavily
X-irradiated allogeneic mice primed with antigenic extracts. If the
ex-
tract immunized the host, then the transferred cells were rejected, and
no sheep hemagglutinins could be detected at the sixth day following
transfer and with simultaneous stimulation of the recipient with sheep
erythrocytes. Maximal stimulation of hosts occurred with a 4-day in-
terval between intraperitoneal cell administration and challenge with
transferred cells. In this assay system, 600 viable bone marrow cells was
the
50
rejection dose
(RD,,,)
or 4000 cells killed by irradiation or
freezing an d thawing, i .e., abou t
a
100-fold difference. Th e RD,, of spleen
cells was lo 3, i.e., abou t the same
as
the activity of spleen cells in
eliciting accelerated rejection. After extensive work with a similar assay
in rabbits, Harris
et
al. (1968) applied their techniques to the assay
of
detergent-extracted antigens.
A
similar system is the transplantation chimera system of Simonsen
and Jensen ( 19 59 ). Transplantation of adu lt cells into im m ature hosts
unable to resist them allows these cells to perpetuate an immune re-
sponse against the alloantigens present in the recipient. On character-
istic of this response is hepatosplenomegaly, which can be quantitated
as the index of enlargement, viz., the spleen weight of organs from in-
jected vs. control animals. The conditions
of
this assay system can
be
adjusted, e.g., b y t i tration of the number of transferred ceIls, SO t ha t
cells derived from a normal donor would not cause splenomegaly.
Preimmunization could thus be measured
as
the splenic index. The op-
posite possibility, of measuring immunotolerance by demonstrating the
inability of the transferred cells to respond to the host’s transplantation
antigen, has been attempted with crude alloantigens without significant
success.
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168
R.
A.
REISFELD AND B .
D. KAHAN
3.
Prolonged
Survival of
Allogmfts
Enhancement.
Flexner an d Jobling (1907) described t he pro-
motion of a transplantable rat sarcoma following pretreatment with
emulsified, heat-k illed tum or cells-the phe nom eno n of imm unological
enhancement. Kaliss ( 1958) showed that humoral antibody mediated
immunological enhancement: sera from animals which had been pre-
treated with certain antigenic preparations were capable of passively
transferring a sta te of altered reactivity to normal recipients (Kaliss an d
Malomut, 19 52). Th e humoral antibody app eared to function by a
central action or immunocompetent cells rather than by a peripheral
combination with antigen (Snell
et
al.,
1960; Uh r an d Baumann, 1961).
The administration of antibody to normal hosts can produce two dis-
pa rate effects on subseque nt grafts-sensitization an d enhancement.
Which one of these two responses predominates depends on several
factors. One factor is the nature of the immunizing stimulus. If t he
stimulus is feeble and fleeting as with killed cells, there is a greater
chance of achieving enhancement than with normal tissue grafts (Kaliss,
1966).
Second, Gorer and Kaliss (1959) suggested that the difference
between immunity and enhancement depended upon the amount of
transferred a ntib od y: 0.5 ml. yielded resistance against a chemically
induced sarcoma, whereas 0.1 nil yielded enhancement, Not only could
the administration of antibody to hosts accelerate the rejection of normal
tissue grafts (St etso n an d Demo poulos, 1958; Siskind and Thomas, 1959;
Chutna and Pokovna, 1961) but also enhancement could be detected
in immune hosts once antibody synthesis had reached its height (Kaliss,
19 52 ), reflecting th e bimodal biphasic imm une response of th e host
(Kaliss, 1966 ). T he thir d factor is th e timing of th e graft challenge. T h e
host immune response became less efficient as the expression
of
t he
delayed-type hypersensitivity component declined ( Batchelor, 19 63 ), in
part du e to t he interference of excess hu m oral antibody w ith th e cellular
expression of delayed-type hypersensitivity ( Voisin and Krinsky, 1961;
Batchelor an d S ilverman, 1 96 2). Conversely, delayed-type hypersensitiv-
ity seemed to predominate during periods of modest antibody produc-
tion, e.g., following X-irradiation (Salvin and Smith, 1959) or after
imm unization with relatively small doses of antige n delivered as eith er
antigen-antibody complexes (Uhr
et
al.,
1957) or into the isolated intra-
dermal site.
In several instances the prolonged survival of normal tissue grafts
has been ascribed to immunological enhancement. The prolonged sur-
vival of ovarian tissue described by Parkes (1958) was probably related
to enhancement or to the nature of the endocrine tissue. Similarily, it
a.
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170
R. A.
REISFELD
A N D
B . D. KAHAN
efficient at releasing th e activity from t he mem branes. About
5
mg. of the
Triton-solubilized m aterial sufficed to kill 50%of the animals, whereas the
residue was essentially inactive. Truly water-soluble material was ob-
tained by digestion of the Triton-soluble water-insoluble fraction with
Crotaleus adamanteus snake venom (E/S = 1 : 4 0 ) . The detergent-
solubilizeii fractions possessed not only enhancing activity but also
homograft-sensitizing potency, confirming the hypothesis that the anti-
genic determinants mediating these phenomena were closely related
(Graff and Kandutsch, 1966).
Zimmerman
et
al.
(1968)
administered subcellular fractions to allo-
geneic hosts in an attempt to prolong canine renal allograft survival.
These transplants had
a
mea n survival time of 10 days. Following p re-
treatm ent w ith three intravenous injections of c ru de nuc lear fraction
over a period of 3 weeks (2 5 mg. antigen tota l), renal allograft survival
was extended to 1 6 days. Hosts treated with more th an 2 5 mg. showed
accelerated rejection. Application of the sonic method of preparation
of transplantation antigens yielded more active fractions. Administra-
tion of 1 o 5.7 mg./kg. of
105,OOOg
cell sa p supe rnate fraction failed to
improve survivals after allografting. However, when 1mg./ kg. of methyl-
prednisolone a nd 2 mg./kg. of azothiaprine were administered daily after
transplantation into a host tha t ha d received 1-2 mg ./kg. of a ntigen in
six doses over a 2-week period, the survival time was dramatically pro-
longed to a mean of 144 days and a maximum survival of 369 days
(Wilson et al., 19 69 a,b ). A significant b ut far less imp ressive prolongation
was obtained by the combination of antigen and antilymphocytic serum.
Wilson (19 70 ) feels that these results ar e be st interpreted as representing
immunological enhancement, because (1 the enhancement is more
obvious after a period of 6-week pre trea tm en t which yields dem onstrable
cytotoxic antibody, (
2 )
discontinuation of the immunosuppressive regi-
men results in prompt rejection suggesting that true tolerance was not
established,
(
3
)
cell-bound complement-fixing antibodies can be detected
in recipient splenic microsomes at t he time
of
transplantation, suggesting
that the host was in the process of an immune response (Wilson and
Wasson, 1965), and
( 4 )
there is a statistical correlation between serum
cytotoxic antibody titers in the recipient and the allograft survival time,
and the antibody titer appears to be related to the antigen dosage and
the time course of administration.
b.
Tobrance.
Th e hypothesis of B urnet and Fenn er (19 49 ) th at
pe rm an en t tolerance to allogeneic tissue could b e established by inocula-
tion of foreign cells into im m atu re hosts was confirmed b y R. D. Owen
(1945) and by Billingham
et
al. (1956a). Immunological tolerance has
been defined as a central failure
of
responsiveness in which immuno-
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TRANSPLANTATION ANTIGENS
171
logically competent cells become unable to initiate synthesis
of
a
restricted range of antibodies following exposure to antigen ( Dresser
and Mitchison,
1968).
Dresser and Mitchison
( 1968)
distinguish three
types of antigens : nonimmunogenic, weakly immunogenic, and strongly
immunogenic materials. Nonimmunogenic antigens paralyze but do not
immunize unless administered in conjunction with adjuvant, e.g., ultra-
centrifugally purified y-globulin ( Dresser, 1961 an d poly-D-amino acids
(Jane wa y and Sela, 196 7). Th e weakly im munog enic antigens im munize
a t
a
dosage greater than that required for paralysis and, thus, show two
discrete zones of immuno logical paralysis (l o w an d high d osa ge ) sepa-
rated by a factor of l o 4 (Mitchison, 196 4). Although a weakly immuno-
genic system had been constructed by testing the response
of
mice to
particular F, hybrid cells (D. R. Bainbridge and Gowland, 1966) or
by
employing donor-recipient com binations differing a t mino r histocompati-
bility loci
(
McK hann, 19 62 ), th e antigens measured in transplantation
are usually of the strongly immunogenic type, i.e., they immunize at a
dose below the tolerogenic level and paralyze at high dosages. The
phenomenon of paralysis with strongly immunogenic antigens was dis-
covered by Glenny and Hopkins (1 92 4) , redefined by Dixon a nd Maurer
(19 55 ), an d reviewed by Smith (19 61) .
Gowland
(1965)
has reviewed th e problem of induction of trans-
plantation tolerance in adult animals. H e has pointed out th at th e genetic
relation between the donor and recipient is a very important factor in
determining the facility of induction
of
tolerance. It is approximately
20
times more difficult to overcome
a
strong H-2 barrier than it is to over-
come a relatively weak H-1, H-3 genetic difference. One might predict
that the application of congenic lines differing at
a
relatively restricted
num ber of stron g specificities wou ld simplify th e induction of transplan ta-
tion tolerance based
upon
the findings of
J.
Klein (19 66 ), viz., when th e
spectrum of H-2 antigen representation was limited, graft survival time
was prolonged. However, Hilgert ( 1967) has observed that, although
such an H-2 system with prolonged first-set graft survival is barely dis-
tinguishable from a weak genetic system
on
the basis of the MST, it does
show distinct immunological properties. Although the administration of
5
x los cells yielded permanent tolerance in some animils differing at
weak genetic loci, pretreatment of animals differing at the H-2 locus
with the same number of cells uniformly yielded immunity.
Tolerance
has
been readily established to the weak male Y-chromo-
soma1 antige n by a single injection of viable cells (M ar ian i et al., 1959)
or with
a
variety of tissue homogenates (Lincler, 1961; Martinez e t al.,
1963; Harvard et al., 1964; K elly et al., 196 4). Tolerance across th e H-1,
H-3 or thc H-3 difference alone could be established
by
parellteral
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TRANSPLANTATION ANTIGENS 173
having mounted an ineffective, a deviated, or an enhancing immune
response. The immune deviation phenomenon of Asherson (1966) may
be
the key to obtaining an ineffective host response. White
et
al.
(1963)
and Wilkinson and White (1966) reported that antigen administered in
incomplete Freund’s adjuvant tended to yield a 7, antibody response,
whereas antigen administered in complete Freunds adjuvant induced a
mixed
y l , yz,
an d delayed-type hypersensitivity response. Asherson
(
1966)
found that preimmunization with aluni-precipitated protein induces only
yl antibody production
upon
challenge with antigen, but immunization
with antigen in complete Freund’s adjuvant induces not only y l r but also
y 2 and delayed-type hypersensitivity responses.
The procurement of true transplantation tolerance will probably
require the application of imm unosuppressive measures, since th e thresh-
old for the induction of immunity with these materials is very low (see
ab ov e) . Based upon the work of Schwartz (19 66 ), metabolic a nd mitotic
inhibitors have been employed to procure prolonged survival. Floresheim
(1967) noted tha t 2030%of mice pretreated with donor cells and
methylhydrazine were permanently tolerant to skin grafts. Seifert
et
al.
( 1966) found the 6-niercaptopurine and methylprednisolone synergized
with antigen to prolong renal allografts in dogs. In the work of Lance
and Medawar (1969) and A. Monaco and Franco (1W9), ant i lympho-
cytic serum (Woodruff and Anderson, 1963) appeared to synergize with
donor antigen p retreatment in prolonging allograft survival. O th er im-
munological procedures yielding prolonged survival include thymectomy
( Miller and Osboa, 1967), radiation ( Taliaferro, 1957), chronic thoracic
du ct fistuIa (McGregor and Gowans, 1964 ), administration of ,,-globulin
( Kamrin, 1959; Mow bray an d H argrave, 19 66 ), an d nonspecific paralysis
by antigenic competition ( Adler, 1964; Liacopoulos and Perramant,
1966), and corticosteroids.
The ability of an extract to induce permanent survival is a critical
test of its antigenic composition, since graft survival demands that all
of the disparate antigenic determinants between donor and host are
present in the extract. On the other hand, immunization can be obtained
even if a relatively small number of antigenic determinants are present
in the material. Thus the tolerance assay has not only an extremely
significant clinical import but also a unique biological significance.
B.
DELAYED-TYPE
YPERSENSITIVITY
ESPONSES
Although the assay systems that employ tissue transplants have a
unique significance in that they directly reflect the
effect
of isolated
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174 R.
A.
REISFELD AND B.
D. K A H A N
antigens on foreign grafts, they are cumbersome and time-consuming.
It
requires at least 2 weeks to complete the administration of antigen, the
skin grafting of the recipient, and the gross and histological inspection
of the test grafts. Furthermore, these procedures are quite wasteful
of
material, since the immunogenic dose of antigen is usually several-fold
greater than the amount needed to detect serological or delayed-type
hypersensitivity ( DTH) activity. In addition, there are probably addi-
tional requirements for immunogenicity as opposed to those necessary
to demonstrate antigenicity in the DTH or serological techniques. It may
be that during purification there are changes in the physical properties
of the antigenic material such that the purified material would contain
less of the more immunogenic material. Dresser believes that imniuniza-
tion actually results from the small amount of aggregated, denatured
bovine y-globulin in commercial preparations of bovine y-globulin.
Furthermore, during purification other contaminant substances which
may function as adjuvants might be eliminated from the preparation, thus
making the purified material less immunogenic than the cruder material,
Although final statements in regard to the actual transplantation activity
of
extracts can only be made after careful grafting procedures, the DTH
and serological techniques may be useful and conserving of antigen. The
application of these techniques is dependent upon the assumption that
these three systems are mediated by the same antigenic determinant or
by distinct determinants which are closely allied in an extract. In crude
mixtures, Brent
et al.
(1962a) found a correlation between the immuno-
genic activity and the inhibition of hemagglutination, and, in relatively
homogeneous preparations of guinea pig transplantation antigen, there
appeared to be a correlation between immunogenic activity and the
ability of the material to elicit DTH responses (Kahan and Reisfeld,
1969a).
A considerable body of evidence supports the hypothesis that trans-
plantation immunity is mediated by a DTH mechanism, i.e., by the
effects of cell-bound or cell-dependent antibody. It would, thus seem
theoretically preferable to employ assay systems that depend upon the
immune mechanism which mediates transplantation rejection, rather than
systems reflecting humoral antibody which has an uncertain role in the
allograft reaction, However, the DTH methods are best applied in a
species who can express cutaneous delayed-type hypersensitivity re-
sponses, The guinea pig has been extensively studied in these systems
and is probably the best species for such studies. The mouse, on the other
hand, appears to be markedly less capable of expressing cutaneous DTH,
although Crowle and Hu (1967) have documented these responses to
dextrans. Dekaris and Allegretti (1968) showed a direct reaction upon
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TRANSPLANTATION ANTIGENS 177
The direct cutaneous hypersensitivity reaction has proved to be a
simple, rapid, and sensitive method to discern fractions possessing allo-
antigenic activity in the course
of
the purification
of
the active principal
in cell extracts prcpared by sonication. Delayed-type hypersensitivity
responses were elicited in guinea pigs by challenge of presensitized hosts
with 10 pg. of Sephadex Fraction I or w ith 0 .1 pg. of acrylamide-purified
component 15 (t h e component tha t had induced accelerated rejection of
allografts in dosages of 1 to 3 pg.) . The cutaneous react ions had the
temporal and histological characteristics of delayed-type responses.
Furthermore, direct DTH reactions could be elicited by challenge of
hosts that had been adoptively immunized by isogeneic lymphoid cells
transfers from animals previously immunized with donor-strain skin
grafts (K ah an , 1967; Kahan et al., 196 8a). Similar DTH responses have
been elicited in the human being by challenge of presensitizcd hosts
with peripheral lymphocytes ( Martin
et
al., 1957) .
In contradistinction to the delayed-type cutaneous hypersensitivity
phenomena are the Arthus reactions which can also
be
elicited against
transplantation antigens. Chutna
et
a?. (1961) found that rabbits ini-
munized with whole epidermal cells emulsified in complete Freund’s
adjuvant developed immediate, Arthus-type reactions upon challenge
with dono r-type cells.
A
similar pa ttern of reactivity was no ted in rabb its
known to be producing alloantibody. Guinea pigs immunized with
allogeneic spleen cells emulsified in complete Freund’s adjuvant also
developed Arthus-type reactions upon challenge with homologous soluble
Sephadex Fraction I antigen. Since complete Fr eu nd s adjuvant is known
to direct the immune response toward the production of cytotoxic anti-
body, t he A rthus reactivity probably reflects the ability
of
th e solubilized
antigen to induce several types
of
immune response directed against
cell-bound transplantation antigens.
Arthus reactions wcre elicited by challenge of human beings produc-
ing monospecific alloantisera with purified transplantation antigen solu-
bilized by sonication ( Kahan
et
al., 19 68 b) . These individuals displayed
well-defined, deep erythematous reactions with a violaceous hue and
central discoloration specifically upon challenge with antigen possessing
the specificity against which they were producing alloantibody. This
reaction was employed to detect the active component following dis-
continuous acrylamide gel electrophoresis. The gel was sliced, the cuts
were eluted in pairs, and the eluates inoculated into a panel of donors
some of whom had been preimmunized with donor spleen cells. Figure 6
reveals u ) that reactions wcre only elicited i n preimniunized donors
(RI A, SG H, C A P) an d not in the nonimmunized individuals
(ROC
and
RO T ) a nd ( b ) ha t th c antigenic activity
was
well localized
on
th e go1 at
cuts
9-10
corresponding to R, 0.80.
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178
R. A . REISFELD
AND
B. D . KAHAN
2.
Transfer Reactions
Brent
et
d.
1962a) further demonstrated that the local passive
transfer of lymphocytes into allogeneic skin produced a delayed-type
imm une response-the transfer reaction. They proposed that this reaction
was du e to th e recognition by the im munocom petent cells
of
the foreigp
transplantation antigens either present in the skin at the site or in the
host leukocytes drawn to the site of inoculation. This response has been
employed to assess the antigenicity of cell extracts. Intraderinal third-
party local possive transfers (Metaxas and Metaxas-Buchler, 1955) of
presensitized cells admixed with donor allogeneic antigen into the skin
of a member of the recipient strain permit the sensitized immunocompe-
ten t cells to respond to their homologous allogeneic antigen in isogeneic,
immunologically neutral soil. Although Brent et al. ( 1962b) obtained
only feeble but definitely positive reactions with this method, sonicated
Sephadex Fraction I yielded intense cutaneous reactions (Kahan et al.,
1968a). The admixture of 50 to 100 pg. of this antigen with 10 million
cells produced an inflammatory reaction which progressively increased
in severity over
36
hours. After several days of persistent inflammation,
only an area of alopecia marked t h e site of t h e transfer. T h e alopecia
reflected the necrosis of entrapped host isogeneic hair follicular elements,
analogous to the cytotoxic effects
of
lymphocytes incubated with antigen
upon neutral cellular elements, presumably mediated by release of a
humoral factor (R udd le and Waksman, 1968).
On the other hand, immune react ions could not be induced when
normal lymphocytes were transferred with antigen into isogeneic skin.
Th e failure
of
the normal lymphocyte transfer test in these circumstances
was attributed either to an inherent requirement for cell-bound antigen
to indu ce the normal lymphocyte transfer reaction (Br ent an d M edawar,
1966) or to an insufficient am oun t of a ntigen e mployed in th e studies to
induce a primsry immune response.
Ramseier and Streihlein (1965) have shown that the dorsal skin of
th e irradiated ham ster survives as a n immunologically inert bu t extremely
sensitive milieu for the interaction of immunocompetent cells with
antigen. In this local third-party xenogeneic transfer system, 2 X 10'
lymph node cells obtained from individuals presensitized against allo-
geneic tissue were admixed with 75 pg. of the corresponding donor
antigen (Kahan et
al.,
196 8a). T he cutaneous sites wer e then scored for
the appearance
of
hard nodules. Nodules were only detected at sites
containing sensitized cells and the homologous allogeneic antigen. The
sites that contained soluble antigen and sensitized cells reacted in a
manner similar to those in which normal donor cells were deposited as
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TRANSPLANTATION ANTIGENS 179
antigen to react with sensitized cells. These tests developed a
strong
reaction at 24 hours which rapidly declincd until 96 hours. On the other
hand, admixtures of two normal nonsensitizcd cell populations developed
positive, but weaker, reactions which persisted over a 96-hour period
(Kahan e t d., 1968a).
Although the transfer reactions were able to distinguish specific
alloantigenic activity
in
putative extracts, they have not proven to be
flexible or sensitive methods for the routine screening of antigenic
fractions.
3.
The
In
Vitro Blastic Trunsformntion System
Morphological transformation of lymphoid cells into blastic elements
which are capable of DNA synthesis and mitosis occurs following ex-
posure to phytohemagglutinin ( Nowell, 1960), to staphylococcal filtrate
(L in g and Husband, 19 64 ), to streptolysin S (Hirschhorn et al., 1964 ), to
rabbit antihuman leukocyte antiserum (Grasbeck e t
al., 1964), to allo-
geneic cells (Bain e t
n l .
19 64 ), or to subcellular antigens against which
the cultured cells have been specifically presensitized
(
Mills, 1966).
The transformation
of
lymphocytes obtained from two different donors
admixed in culture-the mixed lymph ocyte culture reaction-presumably
reflects the recognition of foreign transplantation antigens. Some investi-
gators believe that the strength of the mixed culture reaction is directly
correlated with the genetic disparity between two cell donors (Bain and
Lowenstein, 1964; Moy nihan et ctl. , 1965; Bach and Amos,
1967),
a n d
a
mathematical model has been constructed to quantitate the relatedness
coefficient betw een t w o cell donors base d upon th e degrc ? of blastic
transformation in mixed culture ( Alling an d K ahan, 196 9). Ot he r in-
vestigators have not found a correlation between the mixed cell reaction
an d histocompatibility ( Elves and Israels, 1965; Eijsvoogel et al., 1967;
Festenstein,
1966).
Festenstein (1966) has proposed that this lack of
correlation is du e to the interaction
of
two opposite processes related to
histocompatibility, namely, primary immune recognition and allogeneic
inhibition.
The stimulation of blastic transformation of sensitized cells by ex-
posure to their homologous sensitizing antigen has been observed in the
rabbit, guinea pig, an d hum an (L yc cttc and Peaimain, 1963; Mills, 1966;
Oppenhciin e t
al.,
1967; Benezra et
aL,
19 67) , and this imm une respon-
siveness has been demonstrated to depend upon a DTH mechanism
(Silverstein and Gell, 1962; Mills, 1966; Oppenheim et al., 1967) . One
would, therefore, predict that
it
should be possible to isolate the events of
induction and expression of allograft immunity in ~ i t r o y exposure of
inimunocompetent cells to subcellular transplantation antigen.
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180 R. A. REISFELD AND B. D. KAHAN
The first step in this direction was taken in the guinea pig system.
Strain 13, guinea pig, peripheral lymphocytes presensitized against strain
2 skin grafts were admixed with strain 2 water-soluble antigen prepared
by sonication and gel filtration. A t the third day of incubation, tritiated
thymidine pulsing revealed more incorporation into the DNA of pre-
sensitized cells preincubated with allogeneic antigen than into the DNA
of cells incubated with isogeneic antigen. The marked reactivity of
animals presensitized with tissue grafts to this soluble material re-
enforced the observations obtained with the direct reaction, i.e., during
the course of induction of allograft immunity against transplants, hosts
are specifically sensitized against this antigen. There was greater blasto-
genesis evident after th e third day of incubation th an at
the
seventh day,
which is the peak response for mixed cultures of normal lymphocytes.
Primary responses of normal, guinea pig, peripheral Iymphocytes to
allogeneic antigen occurred less regularly ( Kahan
e t al.,
1968a) .
In subsequent work, Viza et al. (1968) added HL-A antigenic frac-
tions prepared by autolytic solubilization to cultures of nonimmune
allogeneic human peripheral lymphocytes. They measured blast trans-
formation only at
the
fifth day, solely employing morphological criteria
of blastogenesis. T h e authors claim ed u p to
2 m
greater degree
of
blasto-
genesis when 200-300 pg. of antigen, containing the HL-A
1,
2,
8
specifi-
cities, w ere incub ated with cells lacking these specificities tha n whe n th e
antigen was incubated with the donor’s peripheral lymphocytes. There
were highly variable, entirely unexplained patterns of stimulation,
dep end ing upon th e donor of the cultured lymphocytes an d indep end ent
of th e results of excellent tissue typing . In a ddition, there was no evidence
that the cells which had been exposed to antigen had either achieved an
immune state or that they had become tolerant after being exposed to
high dosages of antigen, as ha d be en hypothesized by t he authors.
In elegant work, Manson and Simmons (1969) cultured lymph node
cells in the presence of allogeneic, microsomal, membranous lipoprotein
transplantation antigen. There was a 200% ncrease in blastogenesis, as
determined by thymidine pulsing, at the third day. However, because of
th e lack of reproducibility of t h e primary response with th e thym idine
system, the authors cmployed an assay that directly determined the
immune capabilities of the exposed lymphocytes. Allogeneic normal
lymphocytes of the C57BL strain which had been “immunized by
ex-
posure to allogeneic DBA/2 microsomal antigen
in uitro,
killed allogeneic
donor-type DBA/2 spleen cells which were plaque formers in a sheep
erythrocyte hem olytic system.
In summary, the delayed-type hypersensitivity systems offer unique
opportunities for the study of the induction and expression of alIograft
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TRANSPLANTATION ANTIGENS
181
immunity. Although they have had limited application because of the
facility of the serological techniques, they do provide vital information
for our understanding of the physiology of the transplantation antigens.
C. SEROLOGICAL
ECHNIQUES
1.
Hemagglutination
Although transplantation immunity appears to
be
dependent upon a
cellular effector mechanism, humoral antibody is produced in a number
of species following allografting. Initially, Hildemann and Medawar
(1959)
fou nd that crude membranous or nuclear antigenic matter which
readily sensitized allogeneic animals to reject donortype skin grafts did
not specifically absorb hemagglutinins directed against the antigen donor's
cells. Bre nt et d.
1961)
later succced ed in deve loping a serological assay
fo r transplantation antigens based upo n t hc absorption of hem agglutinat-
in g activity as dete cte d in th e assay of G orer a nd Mikulska
(1954) .
Prior
to the developm ent of th e Gorrr-Mikulska assay method, strong aggluti-
nation
of
erythrocytes suspended in saline was only rarely observed, and
the sera showed rather unpredictable behavior upon freezing, presumably
related to the ir transformation to incom plete antibodies Gorer,
1947).
Gorer and Mikulska (1954) found that certain agents that increased the
erythrocyte sedimentation rate, namely, sera from patients with myelo-
matosis, ovarian cyst pseudomucin, and dextran, were effective in
developing hemagglutinating reactions. Thus, dextran and normal human
serum were used to detect the incomplete antibodies of the H-2 system
which dominate hemagglutination reactions. Alloantisera were diluted
in
1%
dextran, and a
1%
rythrocyte suspcnsion was prepared in a
1:2
dilution of inactivated normal human serum. The agglutination was read
microscopically after incubation at 37°C. for 90 minutes.
C ru de A-line subcellular antigen p rep are d by ultrasonication inhibited
the hemagglutinating and cytotoxic activities of CBA anti-A alloantisera
(Bren t
et
nl., 1 361). The alloantisera were exposed to the antigen for
20 to 25 minutes at 25"C., the insoluble material was removed by ultra-
centrifugation, and the activity
of
the absorbed alloantiserum was tested
against apropriate target cells. Because of the strong anticomplementary
activity of the antigenic preparations, Brent et al.
(1961)
fel t that the
agglutination system was p referable to th e lytic system. Antigen extracted
from 15 mg. wet weight of tissuc perceptibly de creased th e hemagglutina-
tion titer, but antigen from 60 mg. was required to abolish all activity.
On the other hand, 500-700 mg. of recipient strain tissue, e.g., CBA line
tissue, was required to absorb the alloantisera, demonstrating
a
tenfold
specificity ratio. Comparisons of the absorptive p ow er of various fractions
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182
R. A . REISFELD AND B. D. KAHAN
with their ability to induce accelerated rejection suggested that
it
was
possible to correlate these activities. Furthermore, Brent et al.
(1961)
show ed th at six injections of material derived from 250 mg. wet weight
of original dono r tissue provoked t he produc tion of hemagglutinins at a
titer of
1
:
1000
to
1
:
4000.
The findings of Brent e t
aE.
were confirmed by
Davies and Hutchison
(1961).
Hemagglutination methods have been applied by Palm and Manson
(
1966).
After three 5-pg. injections of microsomal membrane lipoprotein,
hemagglutinins were detected in the host. Furthermore, mice presensi-
tized with
50 x
l o6 whole spleen cells responded to
1
p g .
of crude
antigen with the production of specific antibody. These investigators
found that 250 pg./ ml. of antige n inhib ited hom ologous alloantibody,
whereas
2
mg./ml. were required for a nonspecific serum.
In a similar fashion, Graff and Kandutsch
(1966)
raised hemaggluti-
nating antibody by immunization with detergent-solubilized lipoprotein.
2. Leukoagglutination
Because the alloantigenic specificities are not well expressed on the
surface of erythrocytes, methods were devised to employ leukocytes as
the target cells. In the initial work of Amos
(1953))
isoantisera, prepared
by inoculation of hosts with allogeneic tumor cells on two occasions
30
days apart, were incubated for 1.5 hours with allogeneic leukocytes
derived from peritoneal exudates. There was good agreement between
the leukoagglutination da ta a nd t h e results of G orer an d M ikulska
(1954)
employing hemagglutination.
T he recent advances in this techn ique by Am os and Peacocke
(1963-
1964)
have been incorporated into antigen assays by a number of
workers ( see below )
.
3 .
Mixed Agglutination
Abeyounis et
al. (1964)
treated strain
929L C3H
mouse fibroblasts
with alloantibody and then demonstrated their cell surface histocompati-
bility antigens with a developing reagent of Group 0 hu ma n erythrocytes
precoated with mouse antihuman serum at subagglutinating concentra-
tions in the fashion described by F aegraeus an d Epsmark ( 1961 )
.
Adsorp-
tion of the marker erythrocytes to the target cells identified those cells
that had complexed alloantibody. Metzgar et al.
(1968)
employed this
method as well as the agglutination technique of Amos and Peacocke
(1963-1964) to assay the
4
and 4b HL-A antigenic determinants present
in detergent-solubilized preparations. In accord with the phenotype of
the donor, they were able to demonstrate specific inhibition at an agglu-
tinating titer
4
times greater than the minimum concentration required
for the detection of antigenic activity (Metzgar et al.,
1968).
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TRANSPLANTATION ANTIGENS 183
D. CYTOTOXICNTBODY
It
is well known tha t following skin grafting or th e intra de rm al injec-
tion of leukocytes, there is the formation of cytotoxic antibody (Phelps,
1937) . Th e method of Gorer and OG orm an (1956) descr ibed for the
assay of mouse cytotoxins
was
applied by Amos et al. (1963b) to
compare the potency of several antigenic preparations. They found that
the antigens inhibited both agglutinating and cytotoxic antibodies but
that they tended to exert a nonspecific interference with agglutination
reactions. Although studies on sera with allospecific agglutinating and
cytotoxic activities (E ng elfr iet, 1966; W alford et al., 1965a,b;
J.
Bodmer
e t
al.,
1966; Zmijewski and Amos,
1966;
Payne
et
al.,
1967) suggested
that both activities could be found directed against the same antigenic
specificity, leukoagglutinins occurred in only a small number of cases
following skin grafting (Colombani et
al.,
1964), compared to the fre-
quent incidence of cytotoxins. Furthermore, cytotoxic methods tend to
have greater sensitivity in the antigen assay and to have less nonspecific
interference than do agglutination techniques and are, therefore, more
generally applied to this problem.
Antibodies mediating cytotoxic effects have been studied by changes
in morphology (Kalfayan and Kidd, 1953; Shrek and Preston,
1955;
Ellem, 1957; Goldb erg an d Green, 1959; Reif an d N orris, 1960 ), motility
( Nossal an d Lede rberg, 1958; Terasaki et al., 196 0), permeability ( Green
et al.,
1959; Green an d Goldb erg, 1960; Terasaki
et aZ.,
1961 a,b), metabo lic
processes ( Flax, 1956; Landschuetz, 1956; Bickis et
al.,
1959), electric
potential across the skin ( Merri l l and Hanan,
1 9 6 Z ) ,
elicitation of
muscular contraction (Feigen et al., 19 61), an d interference w ith cell
division and virus multiplication ( Roizman and Roane, 1961).
One of the most widely applied methods is the use of supravital dyes,
including
the
exclusion of negrosin (Kaltenbach et al., 1958) or eosin
(Hanks and Wallace, 1958) by viable cells. An automated system
(Melamed et al., 1969) employs a spectrophotometer measuring the
absorption of trypan blue by dead cells at 5900.A.
A
decrease in the total
light transmitted through thc system is correlated with the extent of cell
death; conversely, living cells scatter light and cause increased trans-
mission. There are systematic errors related to the presence of erythro-
cytes which are not affected by these cytotoxic antibodies but yet scatter
light, and to certain cell types, e.g., thymocytes, which are small and
exhibit peculiar optical properties. Amos ( 1969) has employed trypan
blue in a micromethod to detect cytotoxic antibody. In principle this
would afford a more efficient, flexible system than previous assay tech-
niques depending upon trypan blue.
Rotman and Papermaster (
1966)
have demonstrated that fluorescein
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TRANSPLANTATION ANTIGENS 185
Modabber
(
1968 ) who pr ep are d splenic cell extracts following immuniza-
tion to p-galactosidase and d etermined th c content of antibody -producing
cclls by incubating the cell suspension with p-galactosidase. After
thorough washing of the unbound enzyme and addition of the fluoro-
genic substrate, the fluorescent products were measured niicrofluoro-
metrically. This method was believed to be able to detect 100-200 anti-
body molecules and to measure the origin of a response as early
as 12
hours and its persistencc at 6 months after immunization, times at which
fluorescent antibody staining mcthods failed to reveal any antibody.
The interaction of antibody and complement on the cell surface
produces functional 150
A.
holes through which even ribosomes can
escape ( Goldberg and Green, 1960). The denatured cell membrane
components resulting from the antibody's action on the surface of the
target cell are then susceptible to digestion by trypsin, whereas normal
cell mem branes are unaffe cted by this agent. Thus, Hirata (19 63) an d
later Terasaki and Rich (1964) employed differential ccll counting with
a cell particle Coulter counter to detect thc cytotoxic action of allo-
antisera on cells. A sigmoidal curve was obtained with increasing con-
centrations of antibody, permitting application of
a
50 lysis coefficient
with only a 3%error in the range of 24 to 78% ysis. Although the Cou lter
counter method achieved
a
high d eg ree of reproducibility an d objectivity,
this technique had not yet been successfully employed in the inhibition
assays with solubilized histocompatibility substances.
Cytotoxic action of alloantisera can also be detectcd by the loss of
intracellular isotopic markers from labeled cells. The application of
14C-thymidine G. Klein an d Perlmann, 19 63) or j2P (Ellein , 1957; Forbes,
1963) incorporated into DNA showed that the isotope did not leak from
cells exposed to trypsin or deoxyribonuclease but could be released by
heat-killing the cells or by exposure of antibody-damaged cells to trypsin.
T h e radiochromium technique was developed by Goodm an (19 61) to
dem ons trate immune cytolysis of cells othe r tha n erythrocytes in
the
rabbit-antimouse system. Ho we ver, th e blank count of
25%
a t t he 50%
cytolysis point was excessively Iargc. The method was modified by
Sanderson ( 1964, 196 5a,b ), Fifty thousand lymphocyte ta rget cells th at
ha d been intracellularly labeled with sodium dichromate-"Cr were
treated with alloantiserum and guinea pig complement. The cytotoxicity
of the serum was then detected as the
liberation
of chromium in
a
non-
sedinientable form- -the radioactivity prysent in th e sup ern ate of th e
cells was counted a n d appeared to correlate with the cytotoxic activity
of the alloantiserum. Wigzell (1965) found a linear relation between the
uptake of radiochromiuin, suggesting that the uptake per ccll did not
vary with th e cell concentration. H e found th at of the total isotopc tha t
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186 R.
A. REISFELD AND
B.
D. KAHAN
fixed to the cells following five washings, 75% ould be released by the
cytotoxic action of th e alloantisera. H e com pared the isotopic and supra-
vital techniques concluding that the sensitivity
of
the methods was
similar and that the isotopic method might have
a
somewhat smaller
stand ard deviation.
In the chromium assay as employed by Nathenson and Davies (1966),
equal volumes of putative inhibitor are admixed with an antiserum-
complement mixture a nd then 100,000 target cells are ad de d. Th e anti-
serum is employed at 80% ytic death, as measured by chromium release,
which is thus 80 x 75%(chromium fraction x cell death
detected
with
chromium = actual cell de ath ) or abo ut 60%actual cell death. This level
corresponds to less than zero cytotoxicity units, thus representing
a
zone
in which antisera are most vulnerable to nonspecific effects. These authors
judge the ir substance to inhibit an antiserum if i t reduces th e cell dea th
by 30%,.e., in practice by 18%actual cell death, which may be just out-
side the range of
the standard deviation of their method.
In addition, chromium techniques, although offering apparent objec-
tivity, have a lower sensitivity since a large num ber of cells are required
in order to have a sufficient amount of radioactivity incorporated into the
cells.
The development
of
the microcytotoxicity test of Terasaki and
McClelland (1964) represented a real advance in the detection
of
cytotoxic alloantibody. Because this method requires only minute qiianti-
ties of serum and lymphocytes,
it
gains greater than a tenfold increase in
sensitivity over previous techniques. Since the sensitivity of a method is
inversely proportional to the number of target cells employed ( C . Jensen
and Stetson, 1961; Boyse et al., 1962; Wigzell, 1965), an appreciable
increase in sensitivity was obtained by using only
5003000
cells. Micro-
droplets containing 0.003-0.00001 ml.
of
antiserum with 0.003 ml. of
rabbit complement, and 2000 target cells were incubated for
4
hours at
room temperature. The cytotoxic reactions were terminated with 0.002
ml. of 37%ormalin an d th e viability of th e targe t celk assessed by mor-
phological criteria under inverted phase-contrast microscopy. Although
tests employing other techniques had detected cytotoxic alloantibodies
in bu t 6-2m of wom en who ha d had mu ltiple pregnancies (V an Rood
et al., 1959; Payne, 1962; K.
G .
Jensen, 19 62 ), th e Terasaki-McClelland
microdroplet technique revealed that 49% of women having five or more
pregnancies and 16 f women with one to four pregnancies contained
alloantibodies.
In the authors’ hands the inhibition of alloantibody in microdroplets
has proved to be a sensitive, flexible technique for the detection of allo-
antigenic activity. In this assay, 0.001 ml. of alloantiserum diluted at
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TRANSPLANTATION ANTIGENS
187
concentrations of
0, 2, or 4 cytotoxicity units is preincubated with 0.001
ml.
of
serial dilutions of antigen for
1
hour at room temperature. After
this time, 3000 target peripheral lymphocytes and 0.003 ml.
of
rabbit
complement are added and an additional 4-hour incubation performed
at room temperature. The reactions are stopped with 0.001 ml. of 36%
formalin and read under inverted phase-contrast microscopy for the
number of dead cells. Control readings of alloantiserum preincubated
with Hank's bu ffe r alone yield 95%death. Sigmoidal curves of alloanti-
body inhibition are obtained with the addition of serial amounts of
antigen in the preincubation mixture. The inhibitory pattern is specific-
antigenic preparations impair only the activity of alloantisera directed
against antigenic specificities present on the cells
of
the antigen donor.
Thus, in Fig. 5, alloantisera Torino-11.03 and Torino-32.19 are inhibited
by the addition of antigen possessing the corresponding HL-A2 and
HL-A7 determinants, but alloantisera recognizing the HL-A1,3,5,8 anti-
genic specificities are unaffected by preincubation with antigen from the
same donor, who lacks these determinants. The specificity ratio (Sander-
son an d Batchelor, 1 96 8) , i.e., the concentration of antigen req uire d to
inhibit a nonspecific serum vs. that required for the homologous serum,
is a bo ut 300 for th e illustrated alloantigen.
Employing antigens possessing various mosaics
of
the HL-A specifi-
cities to determine the potency and specificity of alloantisera, it has been
possible to characterize three operationally monospecific sera, Torino-
11.03 (anti-HL-A Z), Chayra (ant i-H L-A S), and H utter (ant i-HL-A 7).
In each case the antiserum is only inhibited by the antigenic preparation
possessing the corresponding specificity ( Figs.
7,
8, and 9) .
The specificity ratio of the antibody is then defined as the concentra-
,*-------+ HL-A
f47J
/
TO-11-03 ( o n l i - H L - A 2 )
<.,-,-,
I
x
lo5
z I O ~ 3 x 1 0 ~ 4 x 1 0 ~
5 x 1 0 ~
CELL EQUIVALENTS
Frc.
7. Inhibition
of
cytotoxic reactions
of
Torino-11.03 (anti-HL-A2 typing
antiserum employed at 2 cytotoxic units) by Sephadex Fractions
I
f rom three dif-
ferent cell lines: 0-0
RPMI
1788 (HL-A2,7);
A-ARPMI
7249 ( I IL-A1,2 ,8) ;
1 - 4
P M I
4098
(HL-A3).
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TRANSPLANTATION ANTIGENS
189
antibody but not yet applied for the detection of putative transplantation
antigens may be worthy of investigation. Holm and Perlmann (1967)
and Perlmann
et
al.
(1968) found that the addition of phytohemag-
glutinin or specific antigen to sensitized lymphocytes caused the m to
destroy target cells and that this cytotoxic effect could be measured with
chromate released from the target ccll erythrocyte. These authors further
showed that the lymphocytes of Mantoux-positive individuals were cyto-
toxic for unrelated tissue culture cells following stimulation with puri-
fied protein derivative and that preniixture of these lymphocytes with
allogeneic human lymphocytcs made them cytotoxic for tissue culture
cells. The specificity of thc activated lymphocyte for the target cell is
not d etermined by histocompatibility differences alone, for the re a re some
factors th at control th e reaction a nd dictate the ty pe of target ccll. This
phenomenon might be related to the emission of macrophage-inhibiting
factor from sensitized lymphocytes upon contact with antigen ( Bloom
an d Ben nett, 1966; David, 1966) or to th e release of leukotactic factors
from allogeneic lymphocytes in mixed cultures ( Ramseier, 1967).
V.
Perspect ives
I t has been well docum ented tha t genetic constitution plays a m ajor
role in determining the responsiveness of an individual to an antigen as
dem onstrated b y experiments which indicate th at th e fate of tissue
grafts dep end s upon th e genetic relation between donor a nd host (L ittle
an d Johnson, 1922; Loeb, 1930; Sncll, 19 48 ). T hc large amo unt of experi-
mental work done to investigatc the genetic factors involved in the im-
mune response, in particular the respoiisc of inbred mire and guinea
pigs to syn thetic polypep tide antigens, has been recently revieti ed
(
MC-
Devitt and Benacerraf, 1970).
The ability of these inbred animals to produce antibodies to synthetic
polypeptides was shown to be controlled by an autosomal dominant gene
( McD evitt an d Sela, 19 67 ). It is of interest t ha t in mice it was demon-
strated that this gene (Ir-1) is l inked to one or the other allele of the
H-2
locus which determines the pattern of mouse histocompatibility
antigens. In fact, there is no convincing evidence thus far indicating that
Ir-1 and H-2 arc not identical. Furthermore, it was shown that antibodies
to synthetic polypeptides produced by two inb red strains of mice differed
in their activity to a cross-reacting antigen. This difference in specificity
patterns was determined by a gene linked to
the
H-2
locus
(
McDevitt
an d &la, 19 67 ). Thus, in addition to their obvious role in allograft
rejection and their postulated effects on cell contact and recognition,
histocompatibility antigens may also be of primary importallce in some
of the most basic immunological phenomena.
In this regard, Jerne (1970) has recently proposed some provocative
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TRANSPLANTATION ANTIGENS 191
such as the mode of attachment of histocompatibility antigens to cell
membranes and the cellular origin and rate of their biosynthesis. The
elucidation
of
these phenomena will certainly contribute to an under-
stand ing of t he role that these antigens play in th e mo lecular organiza-
tion of the cell membrane.
Many of these intellectually challenging problems can
be
intelligently
approach ed only aft er biologically an d chem ically well-characterized his-
tocompatibility antigens become available in relatively large quantities.
In this regard, the successful isolation of soluble alloantigens from large
amounts of cells in continuous culture derived from normal human
donors is, indeed, highly promising. Advances made in solubilizing,
purifying, and characterizing these alloantigens are such that i t is now
feasible to obtain them in reasonable quantities. Electrophoretically
homogeneous alloantigens with different specificities solubilized by
physical an d chemical means can now be frag me nted by chemical an d
enzymatic methods to ascertain which portion of the molecule has anti-
genic specificity. Amino acid sequence analyses hopefully will give some
insight into the chemical nature
of alloantigenic determinants and con-
tribute to an understanding of the relationship between the genetic
control of allotypic specificities and the biosynthesis and structure of
alloantigens.
Problems such as the elucidation of the role of histocompatibility
antigens in basic phenomena such as the generation of self-tolerance
and antibody diversity remain a challenge for future research.
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The Role
of
Bone Marrow in the Immune Response
NABIH I. AB DOUl AND MAXWELL RICHTER2
The Harry Websfer Thorp Laboratories. Division o f lmmunochemistry and Allergy.
McGill University Clinic. Royal Victoria Hospital. Montreal. Quebec. Canada
I.
I1
111
IV
.
V.
VI
.
VII.
VIII.
IX
.
X
.
Introduction . . . . . . . . . . . .
A. Abbreviations . . . . . . . . . .
A Brief Survey
of
the Techniques Used for the Detection
of Immunocompetent Cells
. . . . . . . . .
A
. Transfer of Normal or immune Lymphoid Cells to Normal
or
Immunoincompetent Recipients . . . . . . .
B Transfer of Bone Marrow Cells to Immunoincompetent
Recipients
. . . . . . . . . . . .
C
.
Hemolytic Plaque Assay (Localized Hemolysis in Gel) .
E
.
Other
in
Vitro Techniques
. . . . . . . .
Bone Marrow As a Source of Immunocompetent Cells . . .
A Organ of Origin
. . . . . . . . . .
B
.
Differentiation of Bone Marrow Cells
. . . . .
Cells Involved in the Humoral Immune Response
. . .
A
. Antigen Recognition . . . . . . . . .
B. The Macrophage . . . . . . . . . .
C. The Antigen-Reactive Cell . . . . . . . .
D
.
The Antibody-Forming Cell
. . . . . . .
E. Properties That Distinguish the Antigen-Reactive Cells from
the Antibody-Forming Cells
. . . . . . .
F.
Lymphoid Cell Migration in
Vioo
. . . . . .
Cell Interactions Resulting in the Induction of the
Immune Response
. . . . . . . . . .
A
.
Cell Interactions in the Humoral Immune Response
.
.
B
.
Cell Interactions in Cell-Mediated Immune Reactions
. .
C. Postulated Mechanisms of Cell-to-Cell Interactions
.
.
Effects of Irradiation on the Immune Response . . . .
A. Mechanism of Antigen Recognition
. . . . . .
B
.
Graft-versus-Host and Transplantation Rejection Reactions
.
D . Hemolytic Focus Assay . . . . . . .
Cells Involved in Cell-Mediated Immunity
C. The Delayed Hypersensitivity Reaction . . . . .
Cells Affected in Immunological Tolerance
Bone Marrow Transplantation-Application . . . . .
. . . . .
. . . . .
Conclusions
. . . . . . . . . . . .
References
. . . . . . . . . . . .
. 202
. 203
.
203
.
203
.
204
. 205
. 205
. 206
. 207
. 207
.
212
.
213
. 214
. 217
. 222
.
228
. 232
. 234
.
237
.
237
. 242
. 243
.
244
. 246
. 246
. 247
. 249
.
251
. 255
. 257
.
258
Present address: Division of Immunology and Allergy. School of Medicine. Uni-
versity
of
Pennsylvania. Philadelphia. Pennsylvania. 19104
'
Medical Research Associate. Medical Research Council. Canada.
201
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ROLE OF BONE MARROW I N THE
IMMUNE
RESPONSE
203
A. ABBREVIATIONS
The abbreviations used in the text arc as follows:
AFC -antibody-forming cell
( s
ARC -antigen-reactive cell(s )
ASU
-antigen-sensitive units
BGG -bovine ./-globulin
BSA
-bovine serum album in
CFU -colony-forming unit
DNA -deoxyribonucleic acid
GARIG-goat antira bbit imm unoglobulin
GV HR -graft-versus-host reaction
HGG -human ./-globulin
H R B C -horse red blood cells
HSA -human serum album in
K L H -keyhole l impet hemocyanin
M LC -mixed leukocyte cultu re
PFC
-plaque-forming cell(
s )
P P D -purified protein derivative
RFC -rosette-forming cell( s )
RNA -ribonucleic acid
SRBC -sheep red blood cells
1 1
A Brief Survey
o f
the Techniques Used f o r
the Detection o f lrnrnunocornpetent Cells
TRANSFERF
NORMAL R IMM UN E YM PHOID ELLSTO
NORMAL
R
IMMUNOINCOMPETENT
ECIPIENTS
A.
The cell-transfer technique, wherein immune responsiveness is con-
ferred to immunoinconipetent irradiated or tolerant recipients by the
administration of iminunoconipetent lymphoid cells, has been extensively
used for the study of ( 1 ) the kinetics of the primary and secondary
immune responses; ( 2 ) the identity and organ source of the immuno-
competent cells;
3 )
cel l migrat ion pathways, and
( 4 )
the extent of
participation of cells of the donor and recipient animals in antibody
formation. These investigations have centered around the immune
response of th e recipients injected w ith eit he r normal, imm une, or
in vitro
antigen-incnbatcd syngencic
or
allogrncic lymphoid cells
( T.
N. Harris
an d H arris, 1957; T . N. Harris
et
al., 1956, 1967; Saintc-M aric an d Coons,
1964; Gray, 1962; Cochrane and 13ixon, 1962; Makinodan and Albright,
1966; Strober an d M andel, 1969; Miller a nd Mitchell, 1969; Davies, 1969;
Claman an d Chaperon, 1969; Taylor, 1 96 9). Both primary an d secondary
immune responsiveness have been successfully transferred with thoracic
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204
NABIH I.
ABDOU
A N D
MAXWELL RICHTER
du ct cells, spleen cells, lymph n od e cells, thymus cells, an d bon e m arrow
cells, either individually or in combination. Recently, Davies e t
al.
(1966,
1967) demonstrated an antigen-induced
in
vivo
proliferative response
of thymic antigen-reactive cells which was not accompanied by antibody
formation but which was antigen-specific and, therefore, immunological
in nature. It must, therefore, be appreciated that the use of antibody
formation as the sole criterion of th e immun e response permits the detec-
tion of antibody-forming immunocompetent cells only and not of other
immunocompetent cells that are incapable of antibody formation but
which also participate in the immune response.
B.
TRANSFER
F
BONEMARROW ELLS
O
IMMUNOINCOMPETENT
ECIPIENTS
The classic technique utilized to study the iinmunocompetence of
bo ne marrow cells was devised by Till an d McCulloch (1 96 1) . W he n
syngeneic bone marrow cells were injected intravenously into irradiated
mice, macroscopic nodules apparently derived from single cells appeared
in the spleens of the recipients (Till e t al., 1964). These nodules con-
sisted of clones,
the
majority of which were composed either of erythro-
cytes, granulocytes, or megakaryocytes. A small percentage of the clones
were composed
of
mixed populations of cells (Mekori and Feldman,
1965).
The clones retained the capacity to form further colonies if
transferred to other irradiated recipients (Siminov itch et al., 1963) . The
cloning capacity of chromosomally marked bone marrow cells was util-
ized to demonstrate the ability of bone marrow cells to populate the
thymus and spleen (Till e t
al.,
1967) and to show that cells of both the
hematopoietic and immune systems are derived from the same stem cell
( W u e t
al.,
196 8b ). A good correlation was observed between the nu m-
ber of dividing chromosomally marked cells in draining lymph nodes in
response
to
the foot-pad injection of sheep erythrocytes and the number
of plaque-form ing cells de tect ed in vitro ( W u e t al., 196833). However, no
direct chromosone analyses were made
of
the actual plsque-forming
cells. Fractionation of normal bone marrow cells by equilibrium density
gradient ultracentrifugation in BSA resulted in the recovery of a fraction
of nucleated cells which contained up to a thirty-fold greater proportion
of CFU as compared to an uncentrifuged control (Turner e t al., 1967) .
Bone marrow cells were studied extensively by various in vitro techniques
for their myelopoietic potential (reviewed in Piscoitta and Brody, 1968)
an d for their ability to form colonies
in
vitro (BradIey and Siemienowicz,
1968; Iman iura a n d M oore, 19 68 ). Cells responsible for colony formation
in vitro
have, in fact, been found to bc the same cells as those which
for m colonies in th e spleen in v i v o ( W u et
al.,
1968a). The ability of the
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206
NABIH I. ABDOU
AND
MAXWELL RICHTER
antigen-sensitive cells or units an d were show n to proliferate in response
to antigen administration (Syeklocha et al., 19 66 ). A similar m odel
was described by Armstrong and Diener (1969) in which the adminis-
tered lymphoid cells formed colonies in agar that produced antibodies
cap ab le of immob ilizing indicator bacteria.
The work of Playfair
et
al. (1965) has shown that PF C do not belong
to a single clone of antibody -producing cells b u t are derived from m ulti-
ple clones which a re recruited a t different times after immunization. This
conclusion is derived from the demonstration that the development of
PFC occurs in colonies of different sizes in anatomically different sites
in the spleen. Celada and Wigzell (1966) have demonstrated that ani-
mals simultaneously injected with two antigens have antibody-producing
cells against each antigen in discrete anatomical sites in the spleen, thus
demonstrating that the immunocompetent clones which form are antigen-
specific and ar e no t mixed.
E. OTHERn Vitro
TECHNIQUES
The various in
vitro
immunological reactions involving two cell types
have been reviewed in d ep th recently by Coombs a nd Franks (1969).
These reactions depe nd on lattic formation by
the
interacting cells both
of which carry the same antigen resulting in mixed agglutination in the
presence of the antibod y. Examples of such reactions ar e mixed agg lutina-
tion ( Coombs e t al., 19 56 a), mixed antiglobulin reaction (C oo m bs et al.,
19 56 b), and mixed conglutination ( Lachmann
et al.,
1965). Another
group of reactions is that in which sensitization of lymphoid cells with
antibody an d possible uptake of complement results in imm une adherence
(reviewed by Nelson, 1963) or phagocytosis. A third group of reactions
consists of those in which sensitized lym pho id cells interac t with anti-
gens on a target cell , resulting in death of the cell. Receptors on
actively sensitized lymphoid cells (lymphocytes obtained from animals
immunized with foreign erythrocytes or lymphoid cells ) have been
shown to interact specifically with membrane antigens on the foreign
erythrocytes resulting in rosette formation (Biozzi
et
aZ., 1968 ) , or wi th
allogeneic tissue cells in monolayer cultures ( Rosenau and Moon, 1961),
respectively.
Th e correla tion between the R FC and the P FC
is
not clear. Although
Zaalberg
et
al. (1968) found that some RFC could give zones of lysis,
Shearer e t al. (1968) consider that the RFC, the direct PFC, and the
indirect PFC each represents a distinct population of cells. Both RFC
and P FC have been foun d to be lymphoid cells of different sizcs (Storb
an d W eiser, 1967; P. F. Harris et d.,1966) .
T h e induced transformation of a lymphoid cell into a blast cell by
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ROLE OF BONE M A R R O W IN THE IMMUNE RESPONSE
207
antigen in
vitro
has been shown to be immunologically specific (Naspitz
and Richter, 1968; Richter and Naspitz, 1968a; Cowling and Quaglino,
1965;
J.
A.
Mills, 1966; Vischer and Stastny, 1967; Benezera
e t
al.,
1967,
1969) and is probably due to an interaction between the antigen with
its specific receptors on t h e lymp hoid cell (Sell, 196 9). This is probably
followed by the liberation of factors that may result in the further
transformation of other “uncommitted cells (recruitment) (Valentine
and Lawrence, 1969; Dumonde et at., 1969). In t he M L C (Ba i n
et
al.,
19 64 ), receptors on the leukocytes of one of th e donors probably rea ct
with allogenic determinants on th e leukocytes of t h e other d onor resulting
in mutually induced blastgenesis. A factor released by the cells in the
MLC has been shown to
be
capable of inducing blastogenesis in cultures
of cells obtained from only one of the original donors or from an un-
related donor (Kasakura and Lowenstein,
1965;
Gordon and MacLean,
1965) .
Cell suspensions, tissue slices, or organ fragments have been used
in tissue culture systems designed to study the competence of lymphoid
cells (reviewed in D utton, 1 967 ). Imniunocompetence has bee n evaluated
by the degree of antigen-stimulated DNA synthesis (Dutton and Eady,
1964), incorporation of isotopically labeled amino acids into imniuno-
globulin (Fahey
et
al.,
1966) , and
by
other antibody-detection tests such
as phage neutralization ( Saunders and King, 1966).
All the above techniques demonstrate the presence of the precursors
of and/or the actual antibody-forming cells. It is only through the use
of specific markers that the identity of other cell types involved in the
initiation of the immune response can
be
ascertained. The works of
Nossal
e t
al. (1968) utilizing
a
chromosome marker, of MiIIer and
Mitchell (1 96 8) using anti-H, sera in mice, of Lubaroff and Waksman
(1968b) using fluoresceinated antisera in rats, and of Richter and Abdou
(1969) utilizing antiallotype sera in rabbits have greatly clarified the
source, identity, and function of the various cell types engaged in the
immune response.
111.
Bone Marrow As
a
Source of Immunocompetent Cells
A. ORGAN F
ORIGIN
1 .
The
Lymphoid
Cell
Th e source
of
lymphoid cclls in thc various lymphoid tissucs has been
clarified by t h r usc of chromosomally marked bone inarrow cells. These
latter cells, but not labeled lymphoid cells
of
other orga~ls, re capable
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ROLE OF BONE MARROW IN
THE
IMMUNE RESPONSE
209
Bierring a nd G run ne t, 1964a,b). By electron-microscopic studies, lympho-
cytes have been seen crossing the sinusoidal enclothelium of guinea pi g
bone marrow (Hudson
and
Yoffey, 1967).
Bone marrow lymphocytes which are morphologically indistinguish-
able from lymphocytes elsewhere in thc body are different in their de-
velopmental potentialities and function. Whereas peripheral lymphocytes
are unable to protect lethally irradiated mice (Gesner and Gowans,
1962), bone marrow lymphocytes obtained by filtration of bone marrow
through glass wool columns are capable of doing so (Cudkowicz
et
at.,
196413;
P.
F. Harris an d Kugler, 1 963 ). Bone marrow lymphocytes are
probably not end stage cells since they have been shown to undergo
blastogenesis in response to stimulation with phytoh emag glutinin
(
Bishun
et al., 1965; Singhal et al., 1968a; Pegrum et al., 1968) and various anti-
gens (Singhal and Richter, 1968; Singhal et
aZ.,
196813) in vitro.
2. Immunocoimpetent Cells
It has been demonstrated (Tyan and Cole, 1963, 1964, 1965) that
immunocompetent cells are derived from bone marrow or fetal l iver
cells durin g embryo nic life. The so urce of these lymphoid precursor cells
during adult life is not known. Cells obtained from a number of marrow-
derived syngeneic hematopoietic spleen colonies,
upon
transfer to ir-
radiated mice, can establish a large population of inimunocompetent cells
reactive to a variety of antigens. The lymphoid cells of the repopulated
lymphoid organs in these mice are of donor origin as shown by the
presence of the T6 chromosome marker of the donor bone marrow cells
in 100% f the dividing cells in the spleen, mesenteric nodes, and bone
marrow (Trent in et at., 1967) .
The relationship between the bone marrow cells that form colonies
in the spleens in lethally irradiated recipients and the cells that con-
stitute the immune cell system is not well understood. It has been shown
that when chromosomally marked cell suspensions of various adult
lymphoid tissues ar e injected in to lethally irradiated mice, only those cells
derived from th e bone marrow a re fou nd in th c thynius of the host in
significant numbers. Although cells from other lymphoid organs readily
proliferate in the peripheral lymphoid tissue, thcy are rarely found in
the thymus (C.
E.
Fo rd an d Micklem, 19 63). These thymic cells, when
transplanted into other irradiated syngeneic recipients, are capable of
forming spleen colonies an d
of
reconstituting immune competence (Doria
an d Agarossi, 1967, 19 68 ). This latter prop erty can no t be instituted
with
the
transfer of normal thymus cells, These data, therefore, indicate
that the hematopoietic colony-forming cells and the imniunocompetent
cells are derived from the bone marrow stem cells.
That the transplanted chromosomally marked bone marrow cell is
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210
NABIH I. ABDOU AND MAXWELL RICHTER
the cell responsible for antibody production was demonstrated by Wu
e t al.
( 1968a)b). They infused syngeneic chromosomally marked bone
marrow cells into lethally irradiated recipients and observed that the
chromosome marker was found in the cells
of
the hematopoietic colonies
in the spleen, in the thymus, and in the popliteal lymph nodes. Follow-
ing the foot-pad injection of SRBC, more than 50%of the dividing cells
in the regional lymph node carried the donor chromosome marker and
were capable of forming hemolytic plaques in vi tro. On the other hand,
chromosomally marked popliteal lymph node cells
of
mice not given
SRBC did not divide and formed the average number of background
hemolytic plaques.
Further support for the bone marrow origin
of
the antibody-forming
cell is derived from the work of Miller and Mitchell (1968) (see below)
and from in vitro experiments with human bone marrow. Human bone
marrow cells cultured
in vitro
have been shown to secrete immuno-
globulins and antibodies (Lombos e t al., 1963; Van Furth e t al., 1966a).
Bone marrow plasma cells, but not bone marrow lymphocytes, showed
positive fluorescent staining to immunoglobulins IgG, IgM, and IgA.
Furthermore, bone marrow cells of patients with the cold hemagglutinin
syndrome can synthesize cold hemagglutinins in vitro (Van Furth and
Den DuIk, 1966). By combining
in vitro
culture studies and immuno-
fluorescent staining of bone marrow cultures obtained from patients
with multiple myeloma and Waldenstrom’s macroglobulinemia, it was
observed that the plasma cells of the bone marrow can synthesize mono-
clonal immunoglobulins with the same electrophoretic mobility and
the same characteristics of heavy and light chains as the circulating
immunoglobulin (Van Furth
e t
al., 1966b).
Bone marrow cells taken from hyperimmunized mice and rabbits have
been known for a long time to be capable of secreting antibody
in vi tro
(Ludke, 1912; Reiter, 1913; Schilf, 1926). Thorbecke and Keuning ( 1953)
observed antibody production in culture fluids when bone marrow frag-
ments from rabbits immunized to paratyphoid
B
vaccine were cultured
in roller tubes. Several investigators have shown that the antibody re-
sponse can be successfully obtained in the irradiated mouse if the latter
is injected with bone marrow obtained from a hyperimmunized donor
(Hobson
e t al.,
1959; Stoloff, 1960). Lesser amounts
of
antibody are
formed if the genetic strain difference is increased between the donor
and the recipient (Gengozian e t
al., 1961;
Doria et al., 1962; Stoner and
Bond, 1963).
In the adult rabbit, it has been shown that the bone marrow is the
main source of the ARC, both in v h o (Abdou and Richter, 1969a; Rich-
ter et
al.,
1970a) and
in vitro
(Singhal and Richter, 1968). In experi-
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ROLE OF BONE MARROW IN THE IMM UN E RESPONSE
211
ments involving the restoration of immune competence of irradiated adult
rabbits with cells ob t a i n d from a number of lyphoid organs, Richter
e t
uZ .
(1970a) observed that only boric marrow cell suspcnsions could
restore immunocompetence to a variety of antigens. Rabbit lymphoid
cells obtained fro m organs other than the bone m arrow (thy mu s, blood,
appendix, sacculus rotundus, lymph node, and spleen) failed to do so.
Sacculus rotundus cells, mesenteric lymph node cells, and peripheral
leukocytes were able to confer antibody formation in the irradiated re-
cipients with respect to SRBC but not with respect to any of the other
antigens. Evidence has been presented indicating that this response is
probably due to the presence of potential antibody-forming cells in these
organs
as
a
result of prior sensitization with Forssinan antigen or other
antigens which cross-react with SRBC present in bacterial flora
of
t he
gut. The bone marrow of the rabbit, on the other hand, does not con-
tain any antibody-forming cells
(
Richtcr and Abdou, 1969).
3.
The Macrophnge
Th e bone m arrow has becn shown to be the m ain source of precursors
of macrophages in inflammatory reactions and in peritoneal exudates.
Volkman and Gowans ( 196 5), applying 3H -thymidine labeling to the
“skin-window” technique, established that the exudate macrophages in
foci of sterile inflammation are derived from a rapidly dividing pre-
cursor present in the bone marrow. The failure to change the character
of the exudate
by
previous thoracic duct drainage or whole-body X-ir-
radiation accompanied by bone marrow shielding excluded the possi-
bility that invading macrophages could have been derived to any ap-
preciable extent from thoracic duct lymphocytes. Spector
e t
al. ( 1965) ,
using a combination of tritium and colloidal carbon labeling techniques
to label dividing precursors of macrophages, identified the highly phago-
cytic bone marrow-derived circulating monocytes as the antecedent of
the majority of peritoneal exudate macrophages. Further evidence in
favor of the bone marrow origin of the macrophage stems from the work
of Virolainin ( 19 68 ). Radiation chim eras were injected with bone marro w
cells carrying the T6-T6 marker and lymphoid cells from a genetically
different donor carrying a different marker. Peritoneal macrophages as
well as those present in bone marrow, spleen, lymph node, and thymus
of the chimera carried the T6-T6 chromosome marker only. All of
these data indicate that the bone marrow is the main source of macro-
phages and that lymphoid tissues outside the marrow do not contain
precursor cells of macrophages ( Virolainen, 1968; Volkman, 1966; Balner,
1963; Goodm an, 196 4). Furthermore, i t has been demonstrated, using
an in
vitro
culture system, that not only bone marrow cells but also
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212
NABIH I.
ABDOU
AND MAXWELL RICHTER
hematopoietic spleen colonies can give rise to macrophages (Virolainin
and Defendi , 1968).
The origin of macrophages in the visceral organs is still contro-
versial. Pulmonary alveolar macrophages ( Bowden
et
nl., 1969; Virolainin,
1968; Pinkett et
al.,
1966) and liver macrophages (Kupffer cells) (Boak
et al., 1968; Volkman and Gowans, 1965; Kinsky et al., 1969) have both
been shown to b e a t least partly derived from bo ne m arrow precursors. I n
mouse chimeras in which the hematopoietic cell could be identified by
a marker chromosome, it was found that approximately two-thirds of
th e dividing cells in th e lung washings arose from the bone marrow pre-
cursor and one-third were derived from
a
pulmonary parenchymal cell
(Pinkett
et al.,
19 66 ). Still to b e resolved, however, is w he the r th e macro-
phage precursor in the bone marrow is a hematogenous or a lymphoid
cell. Recent findings by Howard et
al.
(19 69 ), Vernon-Roberts (19 69 ),
and Boak et al. (1968) imply a lymphoid cell precursor of the macro-
phage. Although mouse spleen, lymph node, and thoracic duct lympho-
cytes could all apparently give rise to pulmonary alveolar and peritoneal
macrophages in recipient F, hybrids (Howard et al., 19 69 )) t is possible
that
the
responsible lymphocytes transferred could have been bone
marrow-derived. Obviously, our knowledge of the functional hetero-
geneity, origin, and development of the macrophage system
is
too in-
complete to w arrant any fu rthe r serious discussion.
B. DIFFERENTIATIONF BONEMARROW ELLS
The identi ty
of
the bone marrow stem cell responsible for hemato-
poiesis is still unknown. Morphological studies of the developing lymph-
omyeloid tissues of chick embryos have suggested that stem cells may
be blasts with heavily basophilic cytoplasm and a prominent nucleolus
(M oo re an d Owen, 196 7). Studies in rodents have shown tha t mono-
cytoid cells ( Ba rnes and Loutit, 1967a) an d lymphocytes (M offatt et al.,
1967) ar e the pro bab le stem cells. Indirect q uan titative an d morphologi-
cal evidence has shown that the transitional cell in the lymphoid bone
marrow compartment is the hematopoietic stem cell ( Moffatt e t al., 1967) .
During recovery from irradiation, it was noted that the bone marrow
transitional cells decrease in number but not in DNA synthetic activity,
suggesting that these cells are actively dividing and leaving the bone
marrow at a rapid rate.
Stem cell differentiation has been shown to vary according to the
degree of erythropoiesis and granulopoiesis required
of
th e bone marrow.
Suppression of erythropoiesis in guinea pig m arrow results in an increased
lymphocyte content of the bo ne marrow (Osm ond , 196 7). Stimulation
of granulopoiesis, as occurs after the injection of vaccines ( H . pertussis
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213
OLE
OF BONE MARROW IN THE MMUNE RESPONSE
or Staphglococcus aureus) results in an initial rise and then a gradual
drop in the lymphocyte content of the bone marrow (Yoffey, 1955).
Whether the bone marrow stem cell
is
pluripotential or unipotential
is still controversial. By transplanting labeled adult mouse bone marrow
cells into lethally irradiated recipients, evidence has been obtained sug-
gesting that the stem cell can differentiate into any one of
a
number
of cell pathways, with resultant colonies composed of erythropoietic,
myelopoietic, lymphopoietic, or plasmacytopoietic elements (Trentin
et al., 1969). Moreover, transplantation of each type of colony into a
secondary irradiated host can give rise to any type of colony (Curry
et al. , 1967), further attesting to the pluripotential nature
of
the bone
marrow stem cell. Contrary to these findings, Bennett and Cudkowicz
(1967, 1968) showed only a unipotential role for mouse bone marrow
cells by showing that there exist separate progenitor cells for erythro-
poiesis and leukopoiesis. They found no evidence for the shunting of
the transferred bone marrow lymphocytes from or into the production
of nonerythroid cells if they enhanced or depressed erythropoiesis in
recipient mice (Bennett and Cudkowicz, 1968). The differentiation
of the bone marrow stem cell along any
of
the hematopoietic lines
was
shown to be dependent on the microenvironment in which it proliferates
(Wolf and Trentin, 1968). In irradiated mice bearing hematopoietic
transplants, the erythroid-granuloid (
E
:G ) colony ratio in the spleen
was about 3 whether the spleen was kept in situ or transplanted sub-
cutaneously, The E : G colony ratio in the bone marrow stroma was less
than
1
rrespective whether the marrow stroma was in situ or trocar-trans-
planted into the spleen. Dissected erythroid or granuloid spleen colonies
produce all types of colonies upon transplantation, with the E
:
G colony
ratio determined by whether the colonies develop into the spleen or in
the bone marrow. This would indicate that differentiation of stem cells
within the bone marrow is determined by the bone marrow microenviron-
ment and not by the stem cell itself. The nature
of
the inducing factor is
still unknown.
IV.
C el l s I nvo l ved i n t he H u m o r a l Immune Response
In recent years, several cytokinetic models have been described to
illustrate the cellular events in the immune response (Makinodan and
Albright, 1966; Siskind and Benacerraf, 1969; Papermaster, 1967; Sercarz
and Coons, 1963b). In general, these models postulate that when an
immunologically uncommitted progenitor cell
is
stimulated with antigen,
it gives rise to specific
AFC
which differentiate along an irreversible
pathway and, at the same time, produce a new set of progenitor cells
(memory cells) committed to the immunizing antigen. Upon second
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214
NABIH I. ABDOU AND
MAXWELL RICHTER
contact with the same antigen, these memory cells give rise to AFC in
the so-called secondary antibody response as well as to another genera-
tion of memory cells. This scheme probably represents an oversimplifica-
tion of a very complex series of inter- and intracellular interactions. The
recent work of a number of investigators (Miller and Mitchell, 1968;
Davies et al., 1966; Claman et al., 1966; Raidt et at., 1968; Moiser and
Coppleson, 1968; Richter and Abdou, 1969) indicates that the cellular
system responsible for the production of antibodies consists of at least
two and probably three separate cell types. These are the antigen-
processing cell or the macrophage, the ARC, and the AFC.
The various cells that participate in the immune response have been
given different names
by
different investigators:
“X-Y-Z”
by Sercarz and
Coons (1963b), “auxiliary” and “effector” cells by Claman e t al. (1966),
“PC; and “PCC by Makinodan and Albright ( 1966), “Antigen-reactive
cell” (ARC) and “antibody-forming cell” ( AFC) by Miller and Mitchell
( 1968), “antigen-sensitive unit” or “precursor of plaque-forming cell
(
P-
PFC)” by Shearer and Cudkowicz (1969), “reactor cell” by Davies
e t al.
(1966), and “antigen-sensitive cell” by Kennedy et
aZ.
(1965a,b).
Judging from the profusion of terms cited above, it is obvious that
the terminology and the state of knowledge concerning the cells is in a
state of confusion. Cells have acquired names irrespective of their
actual function or the manner in which they manifest themselves in the
immune reaction. Furthermore, the schemes cited above do not take into
account the mechanism of antigen recognition by the immunocompetent
cells-a stage which must necessarily precede that of overt antibody
production-nor do they suggest which cell type might possess the
property of antigen recognition (or, in fact, cognition). The following
sections will, therefore, deal with each of the cell types individually in
order to define better the specific functions and properties of these
cells and to clarify the sequence of intercellular reactions in the immune
response.
A. ANTIGENRECOGNITION
Since the response to antigen is characterized by the extreme specific-
ity of the reaction, it is likely that a subpopulation of lymphocytes reacts
with the appropriate antigen because of the presence of membrane-as-
sociated recognition sites which are structurally complementary to the
antigen (Siskind and Benacerraf, 1969; Mitchison, 1967; Cinader, 1968;
Wigzell, 1969). These sites are considered to be “natural” cell-bound
antibody molecules or the active fragments of the antibody molecules.
The binding of antigen by the cell through its reaction with this anti-
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ROLE
OF
BONE
M A R H O W
IN THE IMMUNE RESPONSE
21s
body receptor probably initiates the sequence of cellular and intcr-
cellular events leading to antibody formation.
There is ample evidence in the li terature indicating the immuno-
globulin nature of these receptors on the surface of normal lymphoid
cells
:
1.
Rabbit peripheral lymphocytes can be stimulated in vitro to trans-
form into blasts by incubation with antiserum directed against allotypic
and other lg determinants
(
SeII a nd
Gell,
1965). Transformation was
shown to b e strictly specific to identifiable allotypic determinan ts.
Chicken spleen lymphocytes were shown to have receptors with
p-chain specificity,
as
demonstrated by their capacity to undergo blasto-
genesis when incubated by rabbit anti-p-chain antiserum, and not with
anti-y-chain antiserum (Skamene and Ivanyi, 1969).
Human thoracic duct and peripheral small lymphocytes show
weak imm unofluorescence for IgM (V an Furth, 1969) and can b e stimu-
lated to transform to blast cells when cultured
in
vitro with anti-IgM
an d anti-IgG sera (V an Fu rth, 1969; Oppenheiin et
aZ.,
196 9). It has also
been d emo nstrated tha t IgG-, IgM -, or IgA-sensitized hu m an erythrocytes
form rosettes with normal peripheral human lymphocytes upon the addi-
tion of the ap pro pria te anti-immunoglobulin seru m (anti-y or anti-p or
anti-a) (Coombs
e t al.,
1969 ). Thus, abou t 10%
of
the peripheral lym pho-
cytes were found to carry immunoglobulin receptors on their surface.
Bert e t
al.
(1968) have demonstrated changes in the physical
properties of normal peripheral lymphoid cells after exposure to species-
specific anti-immunoglobulin serum. A marked reduction in the random
migration of the cells in vitro with reduction of electrophoretic mobility
(Ber t e t
aZ.,
1969) was noted following incubation of human lymphoid
cells with goat antihuman immunoglobulin serum.
Daguillard and Richter (1969) have demonstrated that normal
rabbit lymphoid cells can be induced to transform into blast cells
in
vitro during incubation with goat antirabbit immunoglobulin serum.
Incubation of human circulating lymphocytes with submitogenic
concentrations of rabbit antihuman light-chain antisera suppresses the
mixed leukocyte reaction
in
vito (Greaves et al., 1969) .
The
evidence suggesting that the recognition site on the normal
lymphoid cell surface is, in fact, an antibody or an immunoglobulin frag-
ment with antibodylike activity is based on the finding of Abdou and
Richter (196%) and Singhal and Wigzell (1969) who were able
to
separate norriznl rabbit bone marrow ARC committed to a specific anti-
gen (i.e., H SA ) b y passing the cells through a column of glass beads
sciisitized with the particular antigen ( H S A ) . The ARC retained by the
2.
3.
4.
5.
6.
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216
NABIH
I. ABDOU AND
MAXWELL RICHTER
glass bead column could be eluted from the beads by vigorous shaking.
These cells were found to be capable of undergoing specific blasto-
genesis and responding with increased DNA synthesis upon incubation
with the antigen originally used to sensitize the glass beads, but not
with an y other antigen Singhal an d Wigzell, 1969). Furthermore, th e
eluted cells could transfer antibody-forming capacity only with respect
to the antigen used to sensitize the glass beads and not with respect to
other antigens ( Abdou an d Richter, 1969b).
On
the other hand, the bo ne
marrow lymphocytes which passed through the antigen-sensitized glass
bead column could transfer immunocompetence to all antigens tested
but not to the antigen used to coat the glass beads ( Abdou an d Richter,
1969b).
It was, therefore, concluded that norma1 rabbit bone marrow
lymphoid cells (ARC) possess antibodylike recognition sites on their
surface and that these sites are antigen-specific (Abdou and Richter,
1969b; Singhal a n d Wigzell,
1969).
Investigations conducted with immune lymphoid cells corroborate
the findings with normal cells and strongly imply the presence of an
antibody on the cell surface.
Stimulation of immune cells by hapten-carrier molecules can
be inhibited by prior reaction of these cells with free hapten (Mitchison,
1967; Plotz, 1969; Segal
et
al.,
196 9). Furthermore, such a hapten-immune
cell complex will no t stick to hapten-sensitized glass bead s (M itchison ,
19 67). Similar observations have been repo rted by Naor an d Sulitzeanu
(1969) using BSA as antigen.
Extracts of immune human tonsilar small lymphocytes have been
shown to possess properties of immunoglobulins with immunological
specificity for the antigens to which the donors had previously been
immunized (Merler and Janeway, 1968).
It has been observed that immune rabbit lymphoid cells are in-
capable of transferring specific antibody-forming capacity if they are
incubated with antirabbit immunoglobulin serum prior to their injection
into the irradiated recipient along with the specific antigen. These cells
preincubated with antiimmunoglobulin antiserum also fail to undergo
blastogenesis in responses to stimulation with the specific antigen
in
vitro
(Da gu illard and Richter, 1 970 a). These findings strongly suggest tha t
the antigen an d th e anti-immunoglobulin antibody com pete for th e same
site on th e immunocyte surface a n d that the site is an antibody.
Wigzell and Andersson (1969) were able to purify specifically
directed mouse lymph node AFC by passing them through a glass bead
column sensitized with the original immunizing antigen. All the cells
capable of transferring imniunocompetence with respect to this antigen
were retained by the column, much in the same way as normal ARC arc
1.
2.
3.
4.
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ROLE O F BONE MARROW IN THE IMMUNE RESPONSE
217
retained by an antigen-sensitized glass be ad column ( Abdou an d Richter,
1969b; Singhal an d W igzell, 19 69 ).
5.
Cruc haud an d Frei (1967 ) demonstrated that circulat ing lympho-
cytes obtained from allergic individual form mixed clusters when in-
cubated with SRBC sensitized with the allergen. Clusters were not
formed when unsensi t ized SRBC were incubated with the immune
lymphocytes.
It
has been observed that the concentration of antigenic hapten
required to induce optimal blastogenesis of immune guinea pig lympho-
cytes in u i t r o can be correlated with the affinity of the serum antibodies
produced by these guinea pigs for the hapten (Paul et al., 1967 ). Thus,
the requirement of the cells for
a
high concentration of the hapten in
order to be stimulated to undergo blastogenesis is directly related to
the finding of antibodies of low affinity for the antigen in the circula-
tion of the cell donor, and vice versa.
Biozzi e t al. (1969) found that pretreatment of immune lymph
node cells, obtained from guinea pigs immunized with SRBC, with anti-
sera against guinea pig y-globulin abolished their capacity to form
rosettes in the presence of SRBC in uitro, implying that the antiglobulin
antibody could successfully competc with the antigen for the same site
on th e lymph ocyte surface.
McConnell et a l . (1969) observed that mouse lymph node cells
obtained from mice subsequent to immunization with SRBC could form
rosettes when incubated with the red cells
in
v i t r o . However, rosette
formation could be inhibited by treatment of the immune cells with anti-
heavy-chain antisera. This finding strongly suggests that the specific re-
ceptor on the immune lymphocyte is, in fact, an antibody immuno-
globulin molecule or the ac tive segm ent of the antibody molecule.
However, it must be stated that, in spite of the evidence in favor
of the presence of immunoglobulinlike and antibodylike receptors on the
surface of the immune lymphoid cells, the actual demonstration of anti-
bodies similar in composition to circulating antibodies, either on
or
eluted fro m the
normal
cell, has yet to be reported.
6.
7.
8.
B.
THE
MACROPHACE
The exact site of action of the macrophage in the induction of the
immune response is still obscurc. As will be seen below, two cell types,
the ARC and AFC, have been definitely implicated as participants in
antibody formation. T h e question to be answ ered is not only whether th e
macrophage plays an essential role but
also
where it participates along
the sequence of reactions
and
whether it acts in a
specific
or
a
nonspecific
manner. Is the immune function of the macrophage apparent to the in-
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218
NABIH
I,
ABDOU AND MAXWELL RICHTER
vestigator only because of the artificially contrived nature of the experi-
mental protocols or does it have an immunological role under normal
conditions
in vivo.
Furthermore, if it does have
a
specific role, does its
reaction with the antigen p rec ed e or follow tha t of the ARC ? Is it also
a necessary participant with respect to all antigens as is the AFC?
T h e imp ortance of t h e macrophag e system in th e processing of anti-
gens, with sub seq ue nt release of mo re po ten t immunogenic entities
an d /o r “nonantigenic information” is suggested by th e work of Mitchi-
son (1969 ) and Una nue an d Askonas (19 68) . I n comparing the inimuno-
genicity of free and peritoneal exudate cell-bound forms of protein anti-
gens, such as KLH, HSA, and BSA, it was shown that antigens taken
u p by macrophages, both
in vitro
and
in vivo,
are more potent than th e
native, nonprocessed forms in the in vivo induction of primary immunity
in mice.
W. L. Ford e t at. (1966) observed that rat macrophages which had
ingested SRBC in vivo were capable of transferring specific information
in vitro
to normal rat thoracic duct lymphocytes since these latter cells,
following their separation from the macrophages, could then be trans-
ferred to irradiated immunoincompetent recipients with resultant anti-
body formation directed toward SRBC. Argyris (1967)
has
also ob-
served that macrophages which had ingested antigen (SRBC)
in vivo
could successfully induce specific antibody formation following their
administration into normal syngeneic, but not lethally irradiated, re-
cipients.
The wholly in vitro experiments tend to corroborate the above find-
ings. Cells possessing t he prope rty of sticking to glass, presum ably macro-
phages, are essential for the induction of the primary humoral immune
response in vitro to sheep erythrocytes in the presence of other immuno-
competent nonadherent lymphoid cells (Pierce and Benacerraf, 1969;
Mosier, 1969). Cellfree extracts
of
macrophages incubated with antigen
in vitru are capable of inducing the formation of antibodies in cultures
of syngeneic normal lymphoid cells
in
vitro, some of which may, in fact,
bear the allotype
of
the macrophage donor (Adler et
al.,
1966 ). I t was
also show n that this reaction of antigen with t he ma crophag e precede s
that with the immunocompetent lymphocyte (Fishman, 1961, reviewed
in Fishman,
1969).
Many other investigations have shown that the
RNA fraction(
s )
extracted from peritoneal exudate cells which had been
incubated with the antigen in vitro is immunogenic in vivo and in vitro
(Fishman and Adler, 1963; Askonas and Rhoda, 1965; Gottlieb
et
d.
1967; Feldnian and Gallily, 1967; Unanue and Askonas, 1968; Pinchuck
et al., 1968; Mosier an d Cohen, 196 8).
Bendinelli (1968) induced primary immune response to SRBC with
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ROLE
O F
BONE MARROW IN THE IMMUNE RESPONSE
219
mouse peritoneal exud ate cells a nd observed that th e PFC were, in
fact, lymphoid cells. This finding would appear to run counter
to
that
of
H o l~ ib nd HOUSCY 196 9) w ho observed that inore than
20%
of the
PF C in alveolar exudates from r'ibbit lungs, following th e intrapulmo nary
and intratracheal administration of the antigen
(
SRBC ), were histio-
cytes or monocytes by microscopic and ultrastructural criteria.
There is evidence in thc literature that macrophages obtained from
immune animals possess the capacity to initiate antibody formation when
transferred to normal irradiated recipients. Immune mouse peritoneal
macrophages were cap ab le of transfering inimunocompetence u pon th eir
injection into irradiated syngeneic recipients, with or without the ad-
ministration of antigen, provided th at th e cell donors had be en repeatedly
immunized by the intraperitoneal route ( Kornfeld and Weyzen,
1968).
However, Argyris and Askonas (1968) have observed that within a
population of immune peritoneal exudate cells, the cells not adhering to
glass (lymphoid cells) were shown to be the cells responsible for the
transfer of antibody-forming capscity, thus suggesting that Kornfeld and
Weyzer ( 1968) were, in fact, transferring antibody-forming lymphoid
cells in their peritoneal cell exudate. The findings of Bendinelli (1968)
tend to confirm this suspicion (see a bo ve ). Furthermore, macrophages
are not required for the blastogenic response of imm une cells by antigen
in vi tro (secondary response) ( M. J. Simons and Fitzgcrald, 19 69 ).
The findings presented strongly suggest a role for the macrophage
in the primary, if not in the secondary, immune response. Nevertheless,
the exact role of the inacrophage in the normal induction of the immune
response has been questioned. Moller (1969) observed that normal mouse
peritoneal exudate cells could hemolyze both syngeneic and allogeneic
mouse red blood cells in uitro. In view of the speed of the reaction
(0.51
ho ur ) an d the fa ilure of complement to potentiate th e reaction, i t was
suggested that the reaction was not mediated by antibody and com-
plement but represented a reaction governed by unknown immuno-
logical mechanism, if the reaction is immunologically mediated. This
finding wou ld, therefore, ten d to cast some do ub t on th e significance of
findings of other investigators who have demonstrated that macrophages
are iminunocompetent cells, using the hemolysis in gel technique.
Roelants a nd Goo dman (1 96 9) observed no correlation between the
degree of RNA-antigen complex formation by peritoneal mac rophage s
in vi tro
and the immunopotency of the antigen
i n v i v o .
Although block-
ade of the reticuloendothelial system in mice (Sabet e t
u Z .
1968) and
in rabbits ( A , Cruchaud, 1968) with carbon particles prior to immuniza-
tion resulted in the failure of antibody production, treatment of mice or
guinea pigs with the species-specific antimacrophage serum fails
to
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222 NABIH
I. ABDOU AND
MAXWELL
RICHTER
processed antigen, a messenger RN A, or both, to th e AR C or whe ther
by ingesting and degrading the antigen, they act to decrease the circulat-
ing antigen concentration until the lat ter reaches a n immun ogenic thresh-
old level for the ARC are questions which still await experimental veri-
fication
(
Fishman, 1969; Mitchison, 1 96 9). T he literature do es not help
to clarify the problem but does suggest a well-defined role for the
macrophage, probably in the antigen-processing step and the transfer
of “information” to th e ARC (rev iew ed in C ohn, 1968; Gottlieb, 1968;
Fishman, 1969).
C. THEANTIGEN-REACTIVEELL
The
necessity to invoke recognition of, and interaction with, the
antigen by the immunocompetent cell is basic to the evolution of t he
immune response. The question raised, therefore, is not whether an im-
munocompetent cell must react with antigen but rather whether the
cell that reacts with the antigen is a distinct cell or cell type of which
the sole raison &&re is its ability to interact with the native macro-
phage-processed antigen and to transfer information, in the form of a
highly immunoge nic form of th e antigen, antigen-RNA complex, or just
speci6c RNA, to the AFC; or whether the same immunocompetent cell
processes the antigen and synthesizes antibody.
If
two cell types are
involved, an ARC and an AFC, then the immune system may lend itself
to extensive manipulation since these cells may possess different proper-
ties and may originate in different organs. Such, in fact, appears to be
the
case. Recent investigations in the mouse, rat, and rabbit leave no
doubt as to the dual cell nature of the immune system.
In the mouse, i t has been demonstrated that neither the normal bone
marrow cells nor th e normal thym us cells by themselves can transfer anti-
body-forming capacity with respect to SRBC following their injection
into nonimmu nized, syngeneic, irradiated, imm unoinco mp etent recipients.
However, the recipients of a combination of bone marrow and thymus
cells can respond vigorously with humoraI antibody formation (reviewed
in Miller and Mitchell, 1969; Davies, 1969; Claman and Chaperon, 1969;
Taylor, 1969). Since it has been demonstrated that the thymus cells
must interact with the antigen prior to reaction with the bone marrow
cell (Miller and Mitchell , 1968) and since the bone marrow has been
shown to be the organ of origin of the AFC (Miller and Mitchell,
1968;
Nossal
e t
al.,
1968),
it
would ap pea r that the thymus is th e source of th e
ARC. Further support for this interpretation is provided by the results
of Davies et
al.
(1966,1967) and R. K. Gershon et al. (1968 ) who demon-
strated that thymus cells and not bone marrow cells can respond to
antigenic stimulation with blastogenesis and mitosis, but not with anti-
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ROLE O F BONE MARROW IN THE IMMUNE RESPONSE
223
body formation. Simliar results, demonstrating a necessary interaction
between the thymus and bone marrow cells, have been presented by
Taylor
(1968)
with respect to BSA as antigen.
In th e rat, th e ARC have been fou nd to reside in a num ber of organs,
depending on the type of antigen used and the detector system em-
ployed. Thoracic duct cells from normal donors can restore the primary
immune response to sheep red cells but not to diptheria toxoid in irradi-
ated recipients (Strober and Law , 1969 ). On the other h and, spleen cells
were fo un d to b e ca pa ble of transferring im mu ne responsiveness to
tetanus toxoid ( Strober, 1968, 1969).
W ith respect to the rabbit, i t has been d emonstrated that normal bon e
marrow lymphoid cells, but not lymphoid cells of the other normal
lymphoid organs ( thy m us, spleen, lymph node, sacculus rotunds, a nd
appendix), could
be
stimulated by a variety of antigens
in vitro
to
un-
dergo blastogenesis and mitosis (Sin gha l an d R ichter, 196 8). Fur ther-
more, only normal bone marrow cells could transfer immunocompetence
following their administration into irradiated ( 800 r ) allogeneic recipients
( Abdou and Richter, 1969a; Richter
e t
al., 1970 a). Th e finding that the
antibody-forming cell in the irradiated recipient is of host and not donor
origin (R ich ter an d Abdou, 19 69) is taken as evidence for th e antigen-
reactive nature of the bone marrow cells transferred. The finding that
bone marrow cells obtained from a donor 2 4 4 8 hours following im-
munization (pr im ed bone m arrow ) ar e incapable of transferring anti-
body-forming capacity with respect to the antigen used to immunize the
cell donor but can do
so
with respect to all other antigens tested, in-
dicates that the ARC migrate out of the bone marrow following their
interaction with the antigen and transfer information to the AFC in some
other organ (Ab dou and Richter, 196 9a). Th e bone marrow ARC has
also been shown to be radiosensitive to 800 R total-body irradiation
since they could not be detected in the bone marrow of irradiated
rabbits (Abdou
et
al., 1969) . A good correlation was observed between
th e recovery of th e ARC in th e bone marrow of irradiated rabbits an d
the ability of those animals to synthesize humoral antibodies.
These results demonstrate that the ARC in different species of ani-
mals responsive to the same antigen (SRBC) can
be
normally found
in different organs. They are present in the thymus, spleen, and thoracic
duct in the mouse (Miller and Mtichell, 19 68), in the thymus (Gow ans
and McGregor,
1965)
the thoracic duc t (Strober, 196 8), and spleen
(Strober and Law, 1969) in the rat, b ut only in the b one marrow in th e
normal rabbit (Abdou and Richter, 1969a; Richter et al., 1970a) . The
organ source of the ARC in the other species of animals, including man,
is unknown.
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224
NABIH
I. ABDOU AND
MAXWELL
RICHTER
Knowledge as to the rnono- or pluripotential naturc of the ARC is of
paramount importance in order to elucidate better the cell pathways
concerned with the immune response. The existing evidence indicates
tha t the AR C is a unipo tential cell, tha t is, it is precomm itted, to interact
with a nd be stimulated by one antigen only. Abdou a nd R ichter (196 9a)
have demonstrated that rabbit bone marrow cells obtained
2 4 4 8 hours
following immunization with SRBC failed to confer antibody-forming
capacity with respect to SRBC upon their transfer into an irradiated
(
800
r ) immunoincompetent allogeneic recipent, although the response
of the recipient to a non-cross-reacting antigen, horse erythrocytes
(H R B C ), was of th e same degree as that given by a recipient of normal
bone marrow cells. Thus, only the ARC precommitted to respond to
SRBC vacated the bone marrow following immunization, leaving be-
hind all the ARC directed to HRBC and all other antigens. The rabbit
ARC could also
be
fractionated by passage through an antigen-sensi-
tized (i.e., HSA) glass bead column. All the ARC directed to HSA were
retained by the column, whereas the ARC directed toward all the other
antigens tested were found in the effluent (Abdou and Richter, 1 369b).
Thus, the cells eluted from the column could transfer antibody forma-
tion in an irradiated recipient only to HSA, whereas the effluent cells
could transfer antibody formation to all the antigens tested but not
to HSA. Shearer et al. (1969a,b) hav e also demonstrated the unipotential
nature of the ARC by transferring mouse thymus ARC, at limiting dilu-
tions, and determining the types of immune responses detected. They
concluded that the ARC is unipotent with respect
to
the type of immune
response it will stimulate in the AFC, i.e., direct (19s) PFC, indirect
( 7 s ) PFC, or cluster-forming cells. Talmage et al. (1969) have also
presented sound theoretical arguments in favor of the unipotential nature
of t h e ARC .
Mouse (Raidt et al., 1968) and rat (Haskill, 1969) spleen cells have
been separated into a number of cell fractions, using a density gradient.
The cell fractions that could transfer antibody-forming capacity with
respect to SRBC, either
in
zritro (Ra id t
et
al., 1968) or in viva (Haskill,
1969), were unable to respond to stimulation with
a
non-cross-reacting
antigen.
Ada and Byrt (1969) incubated normal mouse spleen cells with a
highly radioactively labeled preparation of Salmonella adelaide flagellin
antigen. These incubated cells were subsequently unable to confer anti-
body formation with respect to this antigen upon transfer of the cells to
irradiated syngeneic recipients. However, the immune response to other
antigens was normal. The authors present this finding as evidence for
the unipotential nature of the ARC; however, since this antigen, S.
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ROLE OF
BONE
MARROW IN THE IMMUNE
RESPONSE
225
adelaide flagellin, is a thymus- and ARC-independent antigen (see be-
low), it is probable that the cells which were inactivated by interaction
with the labeled antigen were, in fact, unipotent AFC and not ARC.
Trentin
et
al. (1967, 1969) have presented evidence in favor of the
pluripotential n atu re of the ARC. They transferred spleen cells to heavily
irradiated recipient syngeneic mice and observed that the transferred
cells formed discrete clones in the spleens of these mice. They then
transferred cells obtained from these clones in secondary irradiated
recipients whose spleens, containing four to fourteen clones, were now
used to repopulate other tertiary irradiated, iinmunoincompetent re-
cipients. These latter animals responded well to stimulation with a
number of common antigens, thus suggesting that all unipotential ARC
(a n d AF C ) were transferred in th e original spleen cell suspension or
that the ARC is, in fact, pluripotential. A likely possibility is that the
cells passaged through the intermediate irradiated hosts had dediffer-
entiated into stem cells, which may
be
pluripotential in nature, and that
these cells can then give rise to all of the more mature unipotential
ARC in the final recipient.
The majority of the antigens used may
be
considered, at least insofar
as the mouse and rat are concerned, to be thymus-dependent antigens
since they require interaction with
a
thymus cell for the successful in-
duction of the primary immune response. However, since the immuno-
competent thymus cell in the mouse has been shown to be the ARC,
since spleen cells in the mouse can also exhibit ARC activity as they
can successfully substitute for the thymus cells, and since in the rabbit
these same antigens do not require
a
thymus cell for antibody formation
but do require the mediation of a bone marrow ARC, it would appear to
be more scientifically correct and less confusing to refer to the antigens
as ARC-dependent , rather than th ymus -dependen t. Even with respect to
SRBC, the ARC dependency in the mouse is related to the dose of
antigen an d to the strain of m ouse used (Sinclair, 1967; Taylor an d
Wortis, 1 96 8) . Th e imm unc response in the m ouse to S. adelaide flagellin
antigen (Armstrong
e t al.,
1969) and to KLH, horse ferritin, and pneu-
mococcal polysaccharide (Humphry e t al., 1964; Fahey et al., 1965) are
thymus-independent and, therefore, may be considered to
be
ARC-in-
dependent
as
well. Similarly, the immune responses in the rabbit to the
7-globulin in GARIG serum (Daguillard and Richter, 1970b) and to
KLH (Richter,
1970)
are bone marrow- or ARC-independent. These
antigens, therefore, appear to be capable of directly interacting with the
AF C without th e prior intervention of either th e macrophag e or the ARC .
These ARC-independent antigens may bc considered to possess in-
trinsically
a
high affinity for the AFC, thereby imparting to them opti-
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226
NABIH
I.
ABDOU AND
MAXWELL RICHTER
mum immunogenicity. Other antigens must first be processed by the
macrophage and/or ARC in order to increase their immunogenicity or
affinity for the
AFC
(Richter,
1970) .
Are ARC in the mouse thynius-derived but mature under bone
marrow influence or are they bone marrow-derived and mature under
thymic influence? The data presented do not permit for a clear-cut an-
swer. Experimental data indicating bone marrow influence on thymic
anatomy and physiology are scanty. Thymectomy performed in new-
born mice (Trainin and Resnitzky, 1969) and rats (Corsi and Giust i ,
1967)
resulted in an increase in the number of undifferentiated blasts
and impairment of the capacity of the bone marrow to form clones in
lethally irradiated recipients. Although bone marrow cells can recon-
stitute to normal th e structure an d function of lymphoid-depleted thymus
tissue (Gengozian
et
al., 1957; C . E. Ford and Micklem, 1963), yet the
mechanism by which this effect is mediated is unknown. Recently, Burger
and Knyszynski (1969) described a dialyzable agent in the bone marrow
of mice and rabbits capable of stimulating proliferation of mouse thymic
cells both
in v iuo
and
in
uitro. This finding is of interest in view of the
recent observations (reviewed in Gabrielsen et
al., 1969)
tha t in lymph-
openic hypogammaglobulinemia, which is probably due to a defect at
the stem cell level, th e thymus
is
rudimentary and extremely hypoplastic.
A few weeks following bone marrow transplantation in a child with that
defect (Meuwissen et al., 1969b), the shadow in the thymic region
started to enlarge, probably indicating population of th e thymus by t he
adm inistered bon e m arrow cells an d/ or proliferation of thymus cells
in response to a factor(s) produced by the transferred bone marrow.
There are a number
of
possible mechanisms (Fig.
1 )
whereby the bone
marrow in the mouse might convert an immunoincompetent ARC-defici-
ent thymus to an immunocompetent thymus: ( 1 ) differentiation of some
bone marrow cells into ARC in the thymus which, foIlowing antigenic
stimulation, will ind uc e th e transformation of th e other bon e m arrow
cells into ARC; ( 2 ) differentiation of bone marrow-derived cells into
ARC in the thymus but only in the presence of a functioning thymus or
a thymic factor (ho rm on e?) . T h e existence of a thymic hormone or a
factor produced by th e thymus which acts as a hormone has already been
demonstrated and has been shown to be capable of restoring immuno-
competence in an otherwise immunoincompetent thymectomized ani-
mal (Osoba and Miller, 1963; Small and Trainin, 1967; Osoba, 1965;
Law and Agnew, 1967; Goldstein et at., 1966; Law et
al.,
1968); ( 3 ) h e
presence of bone marrow cells in contact with thymic reticuloepithelial
tissue may result in the proper microenvironment for thymic cells to be-
come immunocompetent ARC; and ( 4 ) thymic parenchymal cells may
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ROLE OF BONE
MARROW
IN
T H E
IMMUNE
RESPONSE
227
r 1
t
MATURE
ARC
IMMATURE MATURE
ARK ARC
t
/
I
I
\
THYMUS
CIRCULATION
PERIPHERAL
LY MPHOl D
TISSUES
FIG. 1. A hypothetical representation of the organ source(s) and site(s) of
maturation
of the antigen-reactive cells
( ARC
)
.
be induced to transform into ARC under the influence of a trophic factor
(h orm on e? ) released by the bone marrow (Bu rger and Knyszynski,
1969) .
Th e scheme presented in Fig. 1 is highly speculative as
a
great deal
remains to be learned concerning the factor(
s )
responsible for the mat-
uration of the ARC and its subsequent participation in the immune
response. The scheme presented takes into account only the available
data concerning the situation in the mouse in which a number
of
well-
conducted investigations have unequivocally demonstrated the thymic
origin of the mature ARC (rev iew ed in Miller and M itchell, 1969;
Davies, 1969; Claman and Chaperon, 1969; Taylor, 1969). However, it
must he appreciated that th e immature ARC m ay originate in a different
organ and that i t must mature in the thymus or under the influence of
a thymic trophic factor. Furthermore, the organ of origin and habitation
of the ARC may be different in other species of animals. Experiments in
the rabbit suggest that the mature ARC are found in the bone marrow
(Singhal and Richter, 1968; Abdou a nd Richter, 1 96 9a). W here th e ARC
arise
in
man can only be speculated upon at the present time.
In
summary, the usual ARC-dependent antigen interacts with the
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228
NABIH
I.
ABDOU
AND
MAXWFLL
RICHTER
ARC, inducing them to undergo rapid proliferation and transformation
into lymphoblastoid cells
in
vivo (Davies e t al., 1966; R.
K.
Gershon e t al.,
1968) and
in
v i t r o
(Si ng ha l a n d Richter, 1 96 8) . Adm inistration of vin-
blastine inhibits this response (Syeklocha et al., 1966).Once the ARC are
stimu lated, they vacate their original organ of residence a n d presumably
settle in one of the other lymphoid organs, where they interact with
the AFC or its precursor resulting in antibody formation by the latter
cell (see below ).
D.
THEANTIBODY-FORMINGELL
The role of the AFC in the immune response is indisputable since,
by definition, th e A FC is th e cell th at synthesizes the a ntibody. Ev idence
was present in support of the view that AFC activity becomes manifest
only after th e macrophage-antigen an d /o r th e ARC-antigen interaction
has taken place with the exception of the ARC-independent antigens
which do not require the mediation of the m acrophage an d th e ARC.
The interpretation of these findings is that the AFC responds not to the
native antigen but only to stimulation by a highly processed form of the
antigen the immunogenicity of which is directly related to its affinity
for the AFC (Richter, 1970). The quest ions to be answered, then, are
( a )
What is the organ source of the AFC? and
( b ) s
the AF C a mono-
or pluripotential cell?
There is overwhelming evidence in the literature indicating that
the AFC is morphologically a lymphocyte or a lymphocyte-derived cell
(plasma cell) (Cochrane and Dixon, 1962; Dutton, 1967; Sell and
Asofsky, 1968). Nossal
e t
al. (1967) have shown that lymphocytes from
thoracic duct lymph maintained in single cell cultures can transform
into AFC in vitro, which morphologically appear to be lymphocytes.
MitcheIl and Miller
(Z968a,b)
and Miller and Mitchell (1968) using
anti-H, antiserum, Taylor et
aE.
(1967) using antiallotype serum, and
Nossal et aE. (1968) using chromosome analysis of transferred cells, all
demonstrated that the bone marrow in the mouse provides the AFC . Such
is not the case in the rabbit, however, where it has been definitely shown
using anti-allotype antiserum that the bone marrow does not contain
the AF C (Richter and Abdou, 1969).
Evidence that organs other than the bone marrow in the mouse may
harbou r the AF C has been presented by A rmstrong et al. (1969) . They
transferred cells of normal mouse lymphoid organs along with the anti-
gen SaZn~oneZZuadeluide flagellin into lethally irradiated mice and an-
alyzed the host spleen for its capacity to form foci of bacterial immo-
bilization in vitro. T he bone m arrow, mesenteric lym ph nodes, an d Peyer’s
patches all possessed immunocompetent cells capable of conferring im-
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ROLE
OF
BONE M A R R O W
IN
THE IMMUNE RESPONSE
229
mune responsiveness to the irradiated host. Thus, it must be considered
that all these organs harbor AFC in the mouse.
In th e rat, th e AFC appears to a t least inhab it the thoracic duct a nd
the spleen since both thoracic duct cells and spleen cells have been
found to be capable of transferring antibody-forming capacity to irradi-
ated immunoincompetent recipients
( Strober,
1968, 1969;
Strober and
Law, 1969) .
Although both thymus cells and bone marrow cells are required to
initiate an imm une response in an irradiated mouse, by providing t he ARC
and AFC, respectively, spleen cells by themselves are capable of trans-
ferring antibody-forming capacity
in u iuo (
Claman et
al., 1966;
Kind and
Campbcll, 1968; Stutiiian
et
uZ . ,
1968, 1969) and of initiating antibody
formation in vitro (Pierce and Benacerraf, 1969; Pierce, 1969; Mosier,
1969; Mishell and Dutton, 1967; Marbrook, 1968) indicating that the
spleen contains both ARC and AFC. Although the thesis has been offered
that the spleen contains a single cell type which, through differentiation,
acquires t h e capacity to synthesize antibody following its interaction w ith
the antigen, this view cannot
be
seriously entertained today in view of
the findings that neithcr ARC nor AFC, by themselves, can transfer
antibody-forming capacity to irradiated recipients ( reveiwed in Miller
and Mitchell, 1969; Daviey,
1969;
Taylor, 1969; Claman and Chaperon,
1969) and that the mouse spleen can be fractionated into ARC and AFC
in vitro (Ra id t e t
al.,
1968; Haskill, 1969).
The organ source of the virgin AFC in the rabbit is not known. How-
ever, it is probably not the spleen since exposure of the spleen to
10,000 r X-irradiation, with th e rest of the body lead-shielded, has been
found to result in an enhanccd, rather than in a diminished, immune
response ( Taliaferro and Taliaferro, 1956
) .
Furthermore, the appendix
can also be ruled out as the source of AFC as appendectomy does not
app ear to affect the immu ne response (Sussdorf an d Dr aper , 1956 ).
Is
the AFC a niono- or pluripotential cell? That is, can it synthesize
antibodies directed toward only one antigen or only one antigenic
determinant and need the antibody molecules it synthesizes all be of the
same niolecular class? Attardi et
al.
(1959, 1964),
Nossal
and Makela
19 62) , and Schwartzman ( 1967) have observed, in single cell cultures
of immune lymphoid cells, that though the majority of cells can syn-
thesize antibodies directed to one antigen only, a few cells synthesized
antibodies directed to more than one antigen. However, Makela (1967)
could not detect cells capable of forming antibodies to more than one
antigen using the single cell culture technique.
Playfair
et
al. (1965) have shown tha t PF C
do
not belong to a single
clone of antibody-producing cells but are derived from multiple clones
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232
NABIH
I. A B D O U A N D MAXWELL
RICHTER
arise in some othe r organ a nd m ature in the b on e marrow? Osoba's work
(1968) suggests tha t th e thymic hum oral factor can convert uncomm itted
bone marrow AFC cells into fully competent cells. Heavily irradiated
thymectomized mice grafted with marrow cells and thymus tissue
enclosed in a cell-impermeable chamber could form adequate number of
PFC in their spleens in response to SRBC immunization. Spleen cells of
irradiated thymectomized mice which received only marrow cells failed
to fo rm plaques. T h e kinetics of the splenic PF C response in nonthy-
mectomized marrow chimeras and thymectomized chimeras with thy-
muses implanted under the kidney capsule was identical. Doria and
Agarossi (1967, 1969) and Agarossi and Doria (1968) have shown that
the recovery of th e imm une response to SRBC in lethally irradiated mice
transplanted with either syngeneic or allogeneic bone marrow cells is
conditioned by the host thymus. Donor cells of bone marrow origin in
mitosis were found in the thymus of mouse radiation chimeras. Thymus
cells at different intervals following establishment of the chimeric state
were transferred together with SRBC to lethalIy irradiated syngeneic
mice. Thymus cells from young or syngeneic chimeras were more effective
in transferring plaque-forming capacity than cells from old or allogeneic
chimeras. This would indicate that bo th ARC an d AFC a re present in the
thymus of antigenicalry- nonstimuIated bone niarrow-induced chimeras-
a situation quite different from the normal situation when only ARC are
found in the thymus.
In summary, the bone marrow but not the thymus in the mouse
appears to be the source of the AFC. However, it may not
be
the sole
source. In the rabbit, the organ of origin of the AFC has not as yet been
determined bu t it is definitely no t the bon e marrow. Th e existing evidence
also favors the unipotential nature of the AFC at the stage when it is
actively synthesizing antibodies (AFC,). However, in deference to the
highly controversial and conflicting nature of the results of investigations
concerned with this aspect of the problem, a new scheme and classifica-
tion of th e AFC h as been presented (T ab le I ) w hich incorporates all of
the findings referred to above and which not only makes allowance for,
but, in fact, justifies the various conflicting findings reported to date.
PROPERTIESHAT ISTINGUISHHE ANTIGEN-REAC'IIVEELLS
FROM THE ANTIBODY-FORMINGELLS
On the basis of the investigations cited above, it is obvious that
morphological criteria cannot by themselves be used to distinguish
betw een th e various functionally different cell types, specifically th e
ARC and the AFC, which constitute the immunocompetent lymphoid
cell system.
E.
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ROLE O F BONE MARROW IN THE IMMUNE RESPONSE
233
The following properties can be helpful in distinguishing the ARC
from the A FC:
( a )
organ source, ( b ) sensitivity to irradiation in uivo,
(
C )
sensitivity to irradiation
in
vitro,
( d )
reactivity of immunocompetent
cells toward the native antigen, and ( e physicochemical properties.
The organ of origin is one criterion that distinguishes the ARC from
the AFC in both the mouse and the rabbit . In the mouse, the primary
organ of origin of the ARC appears to be the thymus, whereas the AFC
arises fro m th e bone marrow (re vie we d in Miller and Mitchell, 1969;
Davies, 1969; Taylor, 1969; Claman an d Chaperon, 1969). In the rabb it,
the bone marrow constitutes the oiily source of virgin ARC, whereas
the organ of origin of the AFC has not yet been identified, but it is not
the bone marrow (Richter an d Abdou, 196 9).
A second criterion is that of in vivo radiation sensitivity. In th e mouse,
both the ARC and AFC appear to be equally sensi t ivc (Claman and
Chaperon, 1969; Miller and Mitchell, 1969). In the rabbit, the ARC is
sensitive to 800r total-body irradiation, whereas the AFC is not affected
until 1000 r total-bo dy irradiation is app lied ( Abdou et al., 1969) .
The results of in vitro irradiation experiments corroborate the in vivo
findings in the rabbit. The virgin ARC is inactivated if subjected to
4000r irradiation
in vitro
(Ab don an d R ichter, 1970 a), whereas the
antigen-stimulated ARC
(
Abdou and Richter, 1970a) and the AFC
(Da guillar d an d Richter, 197 0b ) are unaffected by this dose
of
irradiation.
A fourth criterion for differentiating the ARC from the AFC is
their
reactivity toward the native antigen. The ARC can be stimulated by inter-
action with the ARC-dependent antigen
in vitro
(Singhal and Richter,
1968) and in vivo (Davies et al., 1966, 1967) to undergo blastogenesis
an d mitosis, whereas the AF C does not react in this fashion. Furth erm ore,
ARC can be “activated”
in vitro
by interaction with the antigen so as to
enable them to transfer antibody-forming capability to irradiated hosts
(A bd ou an d Richter, 1970a, reviewed in Miller a nd Mitchell, 19 69 ). Th e
AFC cannot be triggered off
by
incubation with the native antigen
in vitro and it cannot transfer antibody-forming capacity in the absence
of stimulated ARC, with respect to the ARC-dependent antigens.
Density gradient separation
of
normal and immune rat spleen cells
followed by testing the immune function of the different cell fractions
subsequent to their transfer to irradiated syngeneic recipients has been
used to stu dy th e density distribution of A RC as compared to A FC
(
Haskill, 1967, 196 9). Haskill rep orte d changes in th e density profile
of ARC in rat spleens following antigen (RBC) stimulation. This change
of profile was noted as early as 10 hours after antigen administration.
Th e AFC were foun d to b e less dense than the ARC. Furthermore, splenic
cells directed to interact with SRBC were found to have densities different
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234 NABIH I. ABDOU AND MAXWELL RICHTER
from those reactive with horse or r at re d cells (Hask ill, 1969). Antibody-
forming cells with the same antigenic specificity obtained from different
organs (spleen, lymph nodes, peripheral blood, thoracic duct cells) dis-
played different sedimentation profiles ( Haskill e t at., 1969). Similar
findings were reported by Raidt et al. (1968) who fractionated mouse
spleen cells on albumin gradients and tested the cell fractions for im-
munocompetence in
u i t ro .
F.
Specific depletion of the recirculating pool of lymphocytes in the rat
by thoracic duct lymph drainage results in variable degrees of lymphoid
depletion in the various organs. The periarteriolar lymphoid sheaths of
the spleen and the cortical zones of lymph nodes are markedly depleted
of small lymphocytes, whereas the bone marrow and thymus content of
lymphocytes is not affected (re vie w ed in W.
L.
Ford and Gowans, 1969).
This would indicate that t he bo ne marrow and thymus contain few or no
circulating lymphocytes and that a large proportion of lymph node and
spleen lymphocytes belongs to the recirculating pool. The circulation of
small lymphocytes between the blood an d the peripheral lymp hoid tissues
takes only a few hours and involves a population of nondividing small
lymphocytes with an average life-span
of
several weeks (Gowans and
Knight, 1964). Bone marrow lymphocytes continuously migrate out of
the bone marrow to the peripheral lymphoid tissues. These cells, how-
ever, do not enter the recirculating pool of lymphocytes (C. E. Ford,
19 66). Parrott ( 1967) injected labeled marrow cells into irradiated mice
and showed that the most prominent site of localization of the labeled
small cells is the red pulp of the spleen and not the recirculating traffic
areas
of
the lymph nodes and spleen. Bone marrow cell migration has
been shown to occur in the developing fetus, in the animal recovering
from irradiation, an d in the norm al adu lt animal (Micklem
et
al.,
1968) .
The criticism against all these experiments is the artificial situation
created , namely, irradiation of t he recipients, th e use of parabiosis models,
and the unknown effects of th e rapid intravenous administration of a
large number of cells.
In the norma1 situation, the number of stem cells in the circulating
blood is small. However, they are increased whenever there is a demand
for stem cells, as during the perinatal period (Barnes and Loutit,
1967 a,b). The source of these circulating stem cells has been shown to
be the bone marrow. Following irradiation, a direct relationship was
observed between the number of CFU in the peripheral blood and the
degree of hyperplasia
of
the bone marrow (Barnes and L outit , 1967a,b).
As a consequence of migration of stem cells between dif fere nt com-
LYMPHOIDELL
MIGRATION
n
Viuo
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ROLE
OF BONE M A R R O W
IN
THE IMMUNE
RESPONSE
235
partments of the lymphoid tissues, the stem cells should normally be
detected in the peripheral blood. This has been demonstrated in the
mouse by Popp
e t
al.
(1958 ) and Goodman an d Hodgson (19 62 ), in the
dog by Cavins e t al. (19 64 ), an d in the guinea pig by Malinin et al.
(
1965) .
It is not known whether bone marrow lymphoid cells migrate
to
the
peripheral lymphoid tissues directly or by way of the central lymphoid
organs, namely, the thymus and the gut-associated lymphoid tissues
(bursa hom olog) . C.
E.
Ford ( 1966) showed that chromosomally marked
bone marrow lymphoid cells injected into syngeneic irradiated niicc
migrate to the thymus, spleen, and lymph nodes. The time sequence for
the appearance of these cells in these organs was not reported. In
a
similar study, it was demonstrated that dividing lymphoid cells migrate
from the bone marrow to the lymph nodes via the thymus.
The
journey
requires a period of several weeks during which time the proliferating
cells probab ly underg o maturation an d / or proliferation ( Goldschneider
an d M cGregor, 1968; Liden an d Linna, 1 96 9) . Micklem et al. (1966,
1968) concluded from their studies that the bone marrow lymphocyte
can migrate directly from the bone marrow to the lymph node without
intermediary stops in the central lymphoid organs. Moreover, the same
authors could not demonstrate migration of cells from the thymus
to
the
bone marrow since no labeled donor thymus cells could be detected in
the bone marrow of the recipient.
In contrast to the bone marrow, the thymus in the mouse and rat is
a major site of production of recirculating small lymphocytes (Miller
et
al.,
196 2). Th ere is evidence in th e li terature (Sainte-Marie an d
Leblond, 1964; Murray and Woods, 1964; Matusuyama
et
al., 1966) that
thymus lymphocytes are released into the blood either directly or via the
lymphatics. Following the infusion of tritiated thymidine directly into
the thymus of the adult rat, labeled small lymphocytes were seen leaving
the thymus via the blood and lymphatics and to then localize in those
areas of the lymphoid tissues in which the recirculating cells predominate,
namely, the Peyer’s patches, the postcapillary venules in the cortical
areas of the lymph nodes, and the periarteriolar areas in the white pulp
of the spleen (Weissman, 1967; Linna, 1967; Goldschneider and Mc-
Gregor, 1968) . This migration pattern is not thymus-dependent and can
be altered by prior incubation of lymphocytes with trypsin (Woodruff
and Gesner, 1968).
Little is known about the migratory pathways of t he ARC and AF C
following administration of the antigen. The findings of Abdou and
Richter (196%) suggest that, in the rabbit, the bone marrow ARC
migrates out
of
the marrow following contact with the antigen (F ig . 2 ) .
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236
NABIH I. ABDOU
AND MAXWELL
RICHTER
L
' ACTiVATED ARC
I
in formot ion
PHASE
I1
( 2
o 5
d o v s l
FIG.2.
The different phases and cell interactions in the
immune
response in
the nomial adult rabbit. ( ARC -antigen-reactive cells; AFC-antibody-forming
cells. )
T he time required fo r the emigration of these cells from th e bon e marrow
is directly related to the dose
of
antigen administered (Sin gha l an d
Richter, 1968; Abd ou an d Richter, 19 69 a). Following th e injection of
an app rop riate amount of antigen, the ARC app ear to vacate th e bone
marrow immediately following interaction with the antigen since the
specifically committed ARC cannot
be
detected in the marrow within
8
to 48 hours following immunization (Phase I ) . These specifically
activated ARC probably migrate to the organ(s) conta ining the AFC
precursors to which it probably transfers specific information (P ha se
11),
which probably stimulates this latter cell to differentiate into an AFC.
These cells can only
be
detected in the spleen of the rabbit commencing
5 to 7 days following immunization (Phase 111) after which time they
can be detected in the circulation and other lymphoid organs (Abdou
and Richter, 1 969 a). In the mouse, i t has been observed that t he ARC
activity of the thymus is lost following antigen administration. As will
be
discussed in Section VII, these findings have been presented as evidence
for th e induction of tolerance in this organ. How ever, it may
be
tha t the
ARC migrate out of the thymus following interaction with the antigen,
leaving behind a specifically tolerant thymus.
Th e migratory habits of the AF C a re not clear. Th e work of Ch ape ron
e t
al. (1968) has demonstrated that, following a single intraperitoneal
injection of SRBC, AFC-producing 19 S antibodies were localized to the
spleen and did not recirculate; AFC-producing
7s
antibodies and the
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ROLE O F BONE MARROW IN THE IMMUNE RESPONSE
237
memory cells rccirculated since increasing numbers were localized to
the spleen, thymus, and bone marrow at increasing intervals of time
following immunization.
The initial events following antigenic stimulation appear to be in-
creased stickiness and clumping of sensitized macrophages ( Nelson and
North, 1965) and the redistribution of the recirculating lymphocytes and
their transformation into blasts in the lymphoid follicles of the periph-
eral lymphoid organs (Hall and Morris, 1965; Weissman, 1967; Austin,
1968; W.
L.
Fo rd a nd Gowans, 1969 ). T he role of the recirculating
lymphocyte in the induction of the humoral immune response has been
dramatically displayed by the work of W. L. Ford and Gowans (1969) .
They observed that isolated spleens of lymphocyte-depleted rats, if
stimulated with SRBC, gave no hemolysin response. However, the im-
mune responsiveness of the spleen was restored to normal levels by
perfusing it with circulating lymphocytes. It appears, therefore, that the
continuous migration of lymphocytes provides cells capable of being
locally stimulated by antigen even though the antigen concentration in
the circulation has fallen to subimmunizing levels.
If macrophages perform
an
essential preliminary role by processing
the antigen, then the movement of lymphocytes past the relatively
sessile macrophages in the follicles would enable contact between these
two cell types to take place an d facilitate the transfer of information from
the macrophage to the imniunocompetent lymphocyte, thus initiating
th e sequence of events leading up to antibody formation.
V.
Cel l In te rac t ions Resu l t ing i n t he I nduc t i on of
the Immune Response
A. CELL
INTERACTIONSN THE
H U M O R A L
MMUNE
ESPONSE
Evidence that m ore than two cell types are required for the induction
of th e primary im mu ne response
in v ivo
and
in vi tro
stems from the work
of a number of investigators (Claman et al., 1966; Davies e t ul., 1966;
Miller and Mitchell, 1968; Taylor, 1968; Richter and Abdou, 1969;
Talmage
e t al.,
1969; Du tton and Mishell,
1967;
Mosier, 1967; Pierce and
Benacerraf, 19 69 ). Miller a nd Mitchell ( 1968) observed that viable
syngeneic thymus or thoracic duct lymphocytes could reconstitute to
normal levels the plaque-forming capacity of spleens of neonatally
thymectomized immunoincompetent mice challenged with SRBC. NO
significant immunological response was achieved by giving syngeneic
bone marrow cells, irradiated thymus or thoracic duct cells, thymus
extracts, or yeast. Spleen cells from reconstituted mice were exposed to
anti-H, sera directed against either the donor of the thymus or the
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238
NABIH I. ABDOU
AND
MAXWELL
RICHTER
thoracic duct cells or against the neonatally thymectomized host. OnIy
isoantisera directed against the host could reduce the number of hemoly-
sin-forming cells present in the spleen cell suspensions, indicating that
the AFC are of host origin and not derived from the donor thymus or
thoracic duct lymphocytes. Thymectomized, irradiated recipients were
also used by the same investigators. The irradiated mice were protected
with syngeneic bone marrow for a period of 2 weeks and then injected
with semiallogeneic thoracic duct cells together with SRBC. These mice
produced a greater number of plaques than irradiated mice which
received the same number of thoracic duct cells without bone marrow.
By using chromosomal markers ( T 6 ) in a syngeneic system, Nossal
et al.
(1968)
confirmed the above findings. When lethally irradiated mice were
injected with mixtures of syngeneic thymus and bone marrow cells, one
of which was chromosomally marked, all the AFC were found to be of
bone marrow origin. These investigators postulated that in the mouse,
the thymus or thoracic duct lymphocytes “recognize” the antigen (ARC)
and interact with it, and this latter reaction triggers off the differentiation
of a bone marrow-derived precursor cell to a specific AFC.
Similar findings were also reported by Davies et al. (1966; reviewed
in Davies, 1969). In their system, chromosomally marked mouse radia-
tion chimeras were used. Although it could be shown that thymus-derived
cells responded vigorously by mitosis to antigenic stimulation, these cells
were not capable of antibody production. In contrast, bone marrow-
derived cells in recipients of bone marrow cells did not respond with
mitosis to antigenic stimulation during the first
3
days following exposure
to antigen, but they were capable of limited antibody production. Anti-
body was maximally produced in recipients of both thymus and bone
marrow cells.
Using both the hemolytic plaque assay (Jerne and Nordin, 1963)
and the hemolytic foci assay (Playfair
et
al.,
1965),
Claman
et
al. (
1966,
1968;
reviewed in Claman and Chaperon,
1969)
demonstrated that ir-
radiated mice had
to
be injected with both syngeneic thymus and marrow
cells in order to facilitate an adequate immune response following the
injection of the antigen (SRBC). Living syngeneic thymus cells were
required since sonicated or irradiated mouse thymus cells or living
heterologous ( rat) thymus cells were incapable of transferring immuno-
competence.
Taylor
(1968)
using a protein antigen
( B S A ) ,
also showed that a
mixture of thymus and bone marrow cells has to be given to irradiated
syngeneic mice in order to obtain an immune response. Reducing the
number of each type of cell resulted in a diminished response. Interest-
ingly, the author found that the administration of
BSA
to the donor
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ROLE OF BONE MARROW I N T HE I MM UN E RESPONSE
239
mouse
24
hours before sacrifice resulted in failure of the donor thymus
cells to interact with normal syngeneic bone marrow cells for the induc-
tion of the im mu ne response in an irradiated recipient (revie we d in
Taylor, 196 9). By using a n allotype m arker in m ice, Taylor e t al. (1966)
showed that mouse bone marrow cells are the cells responsible for anti-
body production.
Abdou a nd R ichter ( 1969a) have demonstrated that reconstitution of
immunocompetence in an irradiated ( 800 r ) immunoincompetent rabbit
could be accomplished by the transplantation of allogeneic bone marrow
cells. Lymphoid cells of other organs did not possess this capacity
(Richter e t al., 1970a). However, by the use of specific anti-allotype
serum,
it
was shown that the AFC in the spleens of the irradiated bone
marrow recipients were of host and not donor origin, thus demonstrating
the ARC nature of the transferred bone marrow immunocompetent cells
(Richter and Abdou, 1969). Thus at least two cell types are definitely
implicated in the immune response in the mouse and the rabbit-the
ARC an d the AFC.
It has been observed that a two-cell interaction is necessary for the
successful ind uction of a secondary immune response with immune rabbit
lymph node fragments in vitro (Richter and Singhal , 1970). I t was
found that
a
cell adherent to glass wool, probably a macrophage, was
essential during the initial period (da ys 1 to 5 ) of the in vitro culture. It
could subsequently be removed w ithout affec ting the immune response
by the fragments. Mosier (1967, 1969) has demonstrated that three cell
types-one glass ad he ren t an d two nonadherent-are requ ired for the
in vitro p r i my immune response of mouse spleen cells to SRBC. These
cells form clusters in the presence of the antigen and the clusters are
antigen-specific. On the basis of these findings, a three-cell model of
antibody formation has been presented by Talmage e t al. (1969 ). In this
model an “adherent” cell would bring together two nonadherent cells,
one of which, considered to be thymus-derived, delivers specific informa-
tion to the second cell, considered to be bone marrow-derived, which is
capable of synthesizing antibody. The recent findings of Pierce and
Benacerraf (1969) strongly support this concept. The induction of a
primary immune response to SRBC in vitro by mouse spleen cells in-
volved
a
two-step cell interaction leading to the activation of the AFC.
The first is a macrophage-dependent phase, complete in about 24 hours,
followed by a macrophage-independent phase, complete in the next 24
hours. Plaque-forming cells were identified 48 hours later, the entire
reaction taking
4
days.
In the mouse, both the macrophage an d the AFC a re of bone m arrow
origin, whereas
the
ARC is normally found in the thymus (Table
11).
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240
NABIH I. ABDOU AND
MAXWELL
RICHTER
In the rabbit, on the other hand, the ARC is normally found in the bone
marrow. The organ source of the rabbit macrophage and AFC remain
to be determined (Table
11).
It is postulated that, depending on the
typ e (pa rticu late versus soluble; AR C-dep endent versus ARC -independ-
en t) an d state (agg rega ted versus aggregate-free) of th e antigen, i t may
interact initially with either
the
macrophage, ARC, or th e undifferentiated
AFC to tr igger off the inter- and intracellular events culminating in
humoral antibody formation (Fig.
3 ) .
These interactions may involve th e
transfer of information or highly immunogenic antigen from the macro-
phage to the ARC which, in turn, transfer information to an antibody-
forming cell, AFC,, which is stil l uncommitted with regard to the
specificity of the immune response (Pathway
I )
or the transfer of infor-
mation directly from the ARC to the AFC, (Pa thw ay 11). A third
TABLE I1
MEDIATINGHE
PRIMARY
UMORAL
MMUNE
ESPONSE
TYPES OF
CELLS A N D SEQUENCE
OF
CELLULAR INTERACTIONS
Cells mediating the immune response
Third cellirs t cell Second cell
Func- Func-
Animal Functional Organ
tional Organ
tional Organ
species
t,YPe
source
typen
source t,ypeh source
~
Rabbit Macrophage ? ARC Bone marrow AFC
?
Mouse Macrophage Bone marrow ARC Thymus AFC Bone marrow
ARC-antigen-reactive cell.
* AFC-antibody-formi ng cell.
mechanism of antibody induction (P ath w ay 111) may involve th e inter-
action of the native antigen directly with the AFC,. Pathways I a n d
11
may
be
characteristic of the majority of antigens used. The failure to
induce tolerance with the aggregated form of the ant igen in the adult
animal as compared with the ease of induction of tolerance with
the
aggregate-free antigen (Abdou and Richter, 1970b;
Biro
and Garcia,
1965; Frei et al., 1965) would suggest that the macrophage is bypassed
by the aggregate-free antigen ( Pathway 11) permitting interaction with
the ARC resulting in a tolerant ARC (Ric hter, 197 0). O n the other hand ,
the aggregate form of the antigen must probably be processed by the
macrophage
(
Pathway I ) before it can function antigenically. However,
the ARC reacts with the macrophage-processed antigen not by becoming
tolerant but by becoming an activated immunocompetent cell . Pathway
I11 may b e observed with the thymus (o r AR C)-indep enden t antigens,
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ROLE O F
BONE
M A R R O W
IN THE IMMUNE
RESPONSE
241
P R OLI F E R A T I ON
ANTIGEN
/
' I
m
/ T R A N S
a n d / o r
P R O L I F
/
INFO
1
O F A N T I GE N -C E LL I N T E R A C T I ON S
I,Il, El REPRESENT THREE POSSIBLE SEQUENCES
P R O L I F
ANTIBODY
INFO = T R A N S F E R OF
INFORMATION
FIG.3. The various antigen-cell and cell-cell interactions resulting in the pri-
mary humoral immune response.
(
U-AFC and P-AFC denote
the
undifferentiated and
precursor states, respectively, of the antibody-forming cell, ARC, antigen-reactive
cells. )
such as KLH, Saliizunella ade la ide flagellin, pneum ococcal polysaccharide,
ferritin, and anti-immunoglobulin serum (see Section IV,C). The inter-
action of the antigen with AFC, in any of the three pathways will render
the AFC specific with respect to this antigen (A F C ,) . Furth er differentia-
tion of this latter cell will result in the terminal cell stage, AFC,, which
may be the plasma cell, which synthesizes and/or stores and/or secretes
the antibody molecules.
Several considerations must bc kept in mind before accepting as fact
the synergistic effect of thymus and bone marrow cells in the immune
response. Several antigens have been shown to be thymus-independent
a nd do not require this type of yynergism (see Section IV ,C ). Synergism
has been uniformly observed in the experiments that involved the use of
certain types of antigens (r e d cells an d protein ant ige ns ) and th e transfer
of immunocompetent cells into heavily irradiated recipients. Radovich
et al. (1968) transferred normal or immune spleen cells ( 4 days post-
immunization) along with normal bone marrow cells and the original
antigen, SRBC, to irradiated recipient mice. They also observed an en-
hanced immune response in the animals given the bone marrow and
spleen cells as compared to those receiving spleen cells only. However,
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242 NABIH I.
ABDOU
AND MAXWELL R I C m E R
they interpreted this synergistic effect of the bone marrow-spleen cell
combination to be nonspecific, in the sense that the addition of bone
marrow cells could enhance the immune response by providing hemato-
poietic precursors and, thus, in some way, prevent depletion of the
precursors transferred with the immune spleen cells or, as stated by
Radovich
et at.
(1968), affect in
a
nonspecific way the localization of
antibody-forming cells in the spleen. However, the findings of Radovich
et
al. (1968) do not altogether support this assumption, since the adminis-
tration of normal bone marrow cells with immune spleen cells and the
antigen, SRBC, into irradiated allogeneic mice resulted in a markedly
enhanced immune response as compared to that observed with recipients
of immune spleen cells and SRBC only. On the other hand, the adminis-
tration of normal bone marrow cells along with normal spleen cells and
SRBC did not result in as enhanced a response as that observed with
the transfer of normal spleen cells and SRBC only. An alternative expla-
nation to the “nonspecific” role of the bone marrow offered by Radovich
e t
al. (1968)
might be that the bone marrow is supplying
both
hemato-
poietic and AFC precursors (Table I ) , each of which is capable of
proliferating into cells of the other cell line. In time of stress, such as
after irradiation, proliferation along the hematopoietic and leukopoietic
cell lines will predominate. Since Miller and Mitchell (1967) observed
that interaction with antigen and maturation of the thymic ARC may
take as long as 7 days before the bone marrow AFC participates in the
immune response, it is probable that very few AFC may be available
from the transferred normal bone marrow. However, if the normal bone
marrow is transferred to the irradiated recipient at
a
time following the
ARC-reactive stage [or as Radovich
et al.
(1968) did, by transferring
normal bone marrow cells and immune spleen cells], then mouse bone
marrow stem cells will be stimulated by the activated ARC to differentiate
into AFC rather than hematopoietic cells thus allowing for a more
marked immune response.
Absence of bone marrow-thymus synergism has been observed by
Craddock
et
al.
(1967) using steroid-treated animals as recipients. Their
findings suggest that the irradiated recipient animals, as opposed to
steroid-treated recipients, are depleted of more cell types than are re-
quired for the successful mediation of the immune response and that
one of these cells is probably provided by the transferred bone marrow.
Furthermore, this cell would not appear to be lost in the steroid-treated
animal.
B. CELLINTERACTIONSN CELL-MEDIATEDMMUNE EACTIONS
An
in vitro
model system simulating the
in
viva thymus-bone marrow
interaction has been described by Globerson and Auerbach (1967).
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ROLE O F BONE MARROW IN
THE
IM MU NE RESPONSE
243
Sublethally irradiated mouse spleen organ cultures required the presence
of normal bone marrow cells for lymphopoiesis to occur, which was
enhanced by the presence of thymus cells. Thymus and lymph node cells,
in the absence of bone marrow cells, failed to induce lymphopoiesis in
the irradiated mouse spleen cultures. Splenomegaly secondary to GVHR
was observed when the spleen slices were grown for 2 to
3
days in the
presence
of
thymus tissue but not when grown in the presence of a
variety of o ther t issues (l iver, kidney, an d sp lee n). This thym ic activity
was demonstrated to be mediated by a humoral factor (Trainin et
al.,
19 69 ). W he n spleen slices were exposed t o lethal doses of irradiation,
reactivation of immuncompetence did not occur unless
both
thymus and
bon e m arrow cells were present. Using the s am e
in
vitro
graft-versus-host
system, Umiel et
ul.
(1968) found that embryonic l iver cells or thymus
cells were incapable of inducing splenomegaly.
A
combination of these
two types of cells, however, was successful in inducing splenomegaly.
Based on these findings, a model has been described by Talmage et
ul.
(1969)
in which
it
is assumed that the passively sensitized thymus-
derived cell, which does not adhere to glass in
uitm
(nonadherent cel l )
is responsible for the specificity of the cell-mediated immune reaction
and that i ts presence
in vitro
enhances the interaction between an
immunocompetent adherent cel l and a n immunocompetent bone m arrow
non adh eren t cell , al l of which are requ ired for th e reaction t o ensue.
C.
The
interaction of two or more cell types is presumably req uire d for
the induction of the primary response to certain types of antigens (see
Section IV ,C ). T he interaction of the thym ic ARC with th e bone m arrow
AFC in the mouse is a requisite for the successful induction of the
humoral immune response. How and where does this interaction take
place?
Is
there a need for
a
third cell type? These are two questions that
ha ve not as yet bee n answe red with respe ct to different types of antigens
and different types of immune responses.
If the function of the macrophage is to process a highly imiilunogenic
form of the antigen, how does the latter get transmitted to the ARC,
which is the cell which presumably participates in the immune response
following the macrophage? Either the immunogen is released into the
circulation by the macrophage or i t is transferred directly from the
macrophage to another macrophage or to the ARC. This material may
then be secreted by the ARC into the circulation where i t will interact
with the potential AFC; or the processed antigen may adhere to the
surface of the AR C which itseIf m igrates to the orga n(
s )
containing the
AFC, where i t interacts physically with AFC, resulting in the passage
of specific information
(
Richter, 1970).
POSTULATED ECHANISMS
F
CELL-TO-CELLNTERACTIONS
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244
NABIH
I.
ABDOU AND MAXWELL RICHTER
Cytoplasmic bridges have been observed to occur between rabbit
peritoneal cells
in vitro
(Aronson, 1963) . Unanue
et al.
(1969) have
observed that antigens stick to and remain on the surface of normal
macrophages and speculated that these niacrophage-bound antigen mole-
cules react with specific antibodylike receptors present on the ARC, thus
bringing th e macrophage into close proximity with th e ARC. Sha rp and
Burwell (1960) and Schoenberg e t al. (1964) have, in fact, observed
cytoplasmic connections between macrophages and lymphocytic cells in
immune lymph nodes in vivo. The frequency of such interactions ap-
peared to increase after antigenic stimulation. McFarland et al. (1966)
have described lymphocytes interacting with macrophages by means of a
cytoplasmic projection called a "uropod" in MLC. Maclaurin
(1969)
has demonstrated cytoplasmic bridging between normal macrophages in
culture in the presence of phytohemagglutinin and between immune
macrophages in the presence of the antigen, tuberculin. Richter and
Naspitz (1968b) observed aggregates or rosette formation in long-term
phytahemagglutinin-stimulated cell cultures composed of a cen tral mono-
nuclear macrophagelike cell surrounded by lymphocytes of varying sizes.
VI. Effects of Irradiation on the Immune Response3
The effects
of
irradiation on the immune response, both
in v ivo
and
in vitro, may vary according to th e type, dose, an d rate of irradiation used
and the type of antigen. Although 750r in vivo irradiation does not in-
activate peritoneal exu date cells which hav e already taken u p th e
antigen, a suppressive effect was described if this dose of irradiation was
administered prior to the uptake of the antigen in vivo (Gallily and
Feldman,
1967) .
Macrophages in mice subjected to
200
to 550 r total-
body irradiation are , however, cap ab le of taking u p ingested a ntigen
in vivo (Mitchison, 1967) an d of taking u p an aggregated protein antigen,
BGG,
in vitro
(Pribnow and Silverman,
1967, 1969).
In fact, irradiation
of
macrophages in Vitro at doses up to 50,000r did not prevent their
capacity to engulf opsonized SRBC in vitro (Perkins e t
al.,
1966). Prib-
now and Silverman (1967, 1969) could succesfully transfer specific anti-
body-forming capacity to sublethally irradiated (
550
r ) immunoincom-
petent mice by the transfer of macrophages preincubated in
uitroi
with
the antigen, aggregated BGG. Similar results were reported by H. Ger-
shon and Feldman (1968) in mice with respect to Salmonella parady-
senteria antigen. On the other hand, they (H. Gershon and Feldman,
1968) could not reconstitute the immune response to SRBC in this way.
Taliaferro
et
al.
( 1964)
observed that 500 r total-body X-irradiation
of
a The subject matter in this section has, in large part, been contributed by Dr.
Leo
Yaffe, Chairman, Department of Chemistry, McGill University, Montreal, Canada.
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ROLE OF
BONE
M A R R O W
IN THE
IMMUNE RESPONSE
245
immunized rabbits resulted in inhibition of the hemolysin response
provided the antigen was not given more than
a
few hours following
irradiation. H owever, Abdou an d Richter (19 69a ) observed that 800 r
was the dose of W O rradiation which was required to depress the
immun e response in the normal adu lt rabbit.
Furtherm ore, it would ap pe ar that results of
in vivo
total-body irradia-
tion are not related, in a simple way,
to
results
of in vitro
irradiation.
Kasakura and Lowenstein (1968) and Daguillard and Richter (1970b)
observed th at 4000 r ““Co irradiation
in vitro
was required to inactivate
human and rabbit lymphocytes, respectively, with respect to their
blastogenic response to stimulation with
mitoniycin-C-inactivated
allo-
geneic a nd xenogeneic lymphocytes, an d 6000 r
in
vitro
irradiation was
required to inhibit completely the phytohemagglutinin-induced blasto-
genic rcsponse (Kasaku ra a nd Lo wenstein, 19 67 ). T h e findings of Vann
and Makinodan (1969) that immune mouse spleen cells could still
respond with antibody formation following 10,000 X-irradiation
in vitro
are supported by similar findings m ade by D aguillard an d Richter (19 70 )
with rabbit lymphoid cells. Why such a high dose of irradiation
(4000
and over) is required
in vitro
to inactivate normal, previously non-
stimulated lymphoid cells ( Abdou an d Richter, 1 97 0a), whereas ex-
posure of the animal to
a
much lesser amount
of
i r radia t ion (800r)
(Abdou et al., 196 9) will result in inhibition of t he immune response,
cannot be ascertained at the present time.
It is extremely important, in the intercomparison of effects obtained
as a
result of irradiation, to insure that all the variables have been kept
constant or taken into consideration so that the results are truly com-
parable. Some of the v ariables a re listed b elow.
1.
Units
in which the irradiation has been measured. Results are
quoted in the literature in roentgens, rems, and rads. The roentgen or
“rep”-roentgen equ ivalen t physical-is defined as “the am oun t of radia-
tion that will produce, in 0.001293 gm. of dry air at S.T.P.,
l
e.s.u.
of
charge of positive ions and
1
e.s.u. of negative ions.” This, when multi-
plied by a factor that takes account of the efficiency of the radiation in
acting
on
mam malian tissue, is known
as
th e “rem”-roentgen equivalent
mammalian. For example, X-rays will cause 20 times
as
much ionization
in tissue as will y-particlcs a nd this must be taken into account. T he “ r a d
is the un it most hightly recom men ded b ecause it is defined simply as 100
ergs of energy deposited per gram
of
material and is indepe nde nt of the
medium or particle causing ionization.
2. T y p e of
radiation.
Even though the radiation dose may be measured
in rads and thus
be
seemingly independent
of
the type of radiation, yet
thc type cannot be neglected. For example, 400 rads
of
whole-body
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246 NABIH I.
ABDOU
AND MAXWELL RICHTER
radiation with a-rays will affect only the skin of the animal initially, d ue
to th e low pe netrat ing power of the a-rays. The same dosage with high-
intensity y-rays would produce a genuine whole-body effect. The same
is true if one irradiates with low-intensity X-rays and attempts a com-
parison with an irradiation with high-energy y-rays such as those
from 6oCo.
3. Time
of
irradiation. Care should be taken to see that cumulative
doses are really comparable. For example, a 10-hour irradiation at a
certain dose ra te may, physiologically or chemically, not b e t h e equ ivalent
of a 1-hour irradiation at
10
times the former dose rate.
VII.
Cells
Involved
in
Cell-Mediated
Immunity
A. MECHANISM
F
ANTIGEN RECOGNITION
As was discussed in Section IV,A, the recognition of the antigen by
immunocompetent
humoral-antibody-forming
cells in a nu m be r of animal
species is effected through the interaction
of
th e antigen with an immuno-
globulin molecule or fragment possessing antibodylike properties on the
surface
of
the cell. In the normal unimmunized rabbit, the recognition
of
the antigen is a unique property of the ARC, which is incapable of
synthesizing antibody
(
Richter a nd Abdou, 1 969 ). In t he imm une animal,
antigen recognition is a property of the AFC or memory cell (Wigzell
and Andersson, 1969; Daguillard and Richter, 1970b). The findings in
the normal animal are in keeping with the concept of clonal selection,
which implies that there exist in the normal unimmunized animal cells
precommitted to interact with any type of antigen (Burnet, 1957, 1962;
Jerne, 195 5). Obviously, th e nu m be r of such cells precom mitted with
respect to any particular antigen would have to be small. Such, indeed,
appears to be the case. Results of a number of investigations, with both
rabbits an d mice, place th e nu m be r of specific precom mitted cells ( A R C )
to
be
between
1
per 1,000 to
1
per
50,000
lymphoid cells (Ab dou and
Richter, 1969b; Ada an d Byrt, 1969; Sulitzeanu a nd Naor, 1969; Naor an d
Sulitzeanu, 1967).
A similar mechanism appears to
be
operating in cell-mediated hyper-
sensitivity a nd transplantation immunity. Incubation of h um an circulating
lymphocytes with a submitogenic concentration of rabbit antihuman
light-chain antiserum results in suppression of the blastogenic response
induced by tuberculin and allogeneic leukocytes ( mixed leukocyte re-
action) (Greaves et
d
969).
This
would indicate that the cells par-
ticipating in cell-mediated imm unity possess on their surface a light chain
or an F ab monomer which acts as a receptor site. Talmage
et al.
(1969)
have postulated a model in which they attribute the recognition of the
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ROLE OF BONE MARROW I N THE IMMUNE RESPONSE
247
antigen in cell-mediated immunity to a cytophilic antibody present on
the surface of the reactive cell.
T he clonal selection theory does not, however, ap pe ar to
be
applicable
to an un derstanding of t he cellular events culminating in th e cell-mediated
(
GVHR, delayed hypersensitivity reaction) immune reaction. It has been
demonstrated that the percentage of lymphoid cells capable of inducing
a GVHR or of participating in the MLC-induced blastogenic reaction is
much greater than can be anticipated on the basis of randomized clonal
selection and is much greater than the figures cited above with respect
to the humoral immune response. As many as 1-3 out of every 100
lymphoid cells appear to be capable of initiating and/or participating in
the cell-mediated immune reaction induced by any one specific antigen
(Nisbet et al., 1969; Wilson et
nl.,
1968; Sinionsen, 19 67 ). These
ap-
parently contradictory findings ( humoral versus cellular immunity)
suggest that the cell mechanisms mediating the two types of immune
responses are different, both qualitatively and quantitatively. Richter
e t al. (1970b) have, in fact, demonstrated that the cells in the rabbit
tha t participate in the h ost-versus-graft reaction ar e functionally different
from those that m ediate th e humoral immune response in that the former
response does not require the ARC. It would, therefore, appear that the
ARC is
a
unipotential cell, capable of interacting with one antigen only,
whereas the comparable cell mediating the cellular immune reaction, if
it functions in a manner similar to the AR C in hum oral imm unity, would
appear to be pluripotential.
B. GRAFT-VERSUS-HOSTND TRANSPLANTATIONEJECTIONREACTIONS
Lymp hocytes are th e effector cells in the GVH R reaction (re vie w ed
in Gowans and McGregor, 1965; Meuwissen et al., 196 9a). Dicke et
al.
(
1968,
1969) have conclusively shown, by cell fractionation studies, that
lymphocytes are the cells responsible for the GVHR in the mouse and
monkey. Mouse spleen or monkey bone marrow cells were fractionated
on a discontinuous albumin gradient and the hematopoietic capacity and
GVHR activity of the cell fractions obtained were studied in lethally
irradiated allogeneic recipients. A cell fraction rich in blast cells showed
a ten-fold increase in the concentration of CFU (index of hematopoiesis)
and a more than tenfold decrease in GVHR activity as compared to the
original cell preparation. No secondary disease was observed in recipients
that received this fraction. A second fraction, composed mainly of
lymphocytes, was very
poor
in reconstituting hematopoietic activity but
was very active in inducing GVHR. Shortman and Szenberg (1969),
using fowl peripheral leukocytes, have shown that
a
minor population of
lymphocytes are t he active cells in t he GV HR . Th e active cells, however,
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248
NABIH I. ABDOU AND
M A X W L L
RICHTER
were not found to be physicocheinically homogeneous since they could
be obtained as a series of peaks u po n fractionation in a density grad ient.
The source
of
cells responsible for graft rejection is unsettled.
Although the thymus in the mouse is essential for the induction of
cellular imm unity (Miller a nd Osoba, 19 67 ), its role in the development
of
the GVHR is controversial. R.
L.
Simmons et al. (1965) have demon-
strated that the GVHR induced in lethally irradiated thymectomized
mice with allogeneic bone marrow cells was as intense as the GVHR
induced in nonthymectomized irradiated hosts. Field and Gibbs ( 1965) ,
however, have shown that thymectomy increases the susceptibility of F,
hybrid rats to GVHR induced by the intraperitoneal injection of parental
strain spleen cells. Although spleen cells taken from normal donors were
shown to be effective in in du cin g GVH R, spleen cells taken from a du lt
mice which had been thymectomized at birth could not induce this
reaction, suggesting a thymic origin for the G VHR cell (Da lma sso et
al.,
19 63 ). In c ontrast, ablation of th e bursa of Fabricius in the chicken does
not decrease the capacity of the circulating lymphocytes to exert a
GVHR (Warner , 1965).
The results of other investigations are conflicting since it has been
observed tha t thymocytes are bo th highly effective (M . W . C ohen
et
al.,
1963; Stutman
e t
al.,
1968 ) an d poorly effective (Billingham a n d Silvers,
1964 ) in inducing GVH R in appropriate recipient hosts. In systems where
thymus cells were fo un d to be effective in inducing GVH R, no synergism
was observed between thymus and bone marrow cells since the addition
of bone marrow cells to thymus cells did not render the latter more
efficient in inducing GVHR in the F, host (Stutman and Good, 1969).
However, results of an opposite nature demonstrating bone marrow-
thymus synergism have recently been obtained by Argyris ( 196913).
Willard and Smith (1966) studied the capacity of syngeneic trans-
planted mouse lymphoid cells to reject allogeneic bone marrow cells in
irradiated recipients. The following decreasing order of effectiveness of
the transferred cells was observed: leukocytes, lymph node cells, spleen
cells, and peritoneal exudate cells. Thymocytes and marrow cells were
not effective. The failure of the syngeneic bone marrow to reject the
allogeneic marrow in the lethally irradiated mouse could have been due
to proliferation of the hematopoietic cells in the transferred syngeneic
marrow along erythropoietic cell lines rather than along immunoconipe-
tent cell lines or to the absence of mature immunocompetent cells capa-
ble
of
mediating the GVHR reaction. Although bone marrow cells by
themselves ar e incapab le of in du cin g th e GVHR (Billingham an d Silvers,
1964; Stutman and Good, 1969; Dicke et al., 1969) the lymphoid cells
that ar e capable of inducing GVHR have been shown to b e d erived from
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ROLE OF BONE MARROW I N
THE
IMMUNE RESPONSE
249
bone marrow ( Goldschiieider and McGregor, 1968; McGregor, 1968;
Ty an an d Cole, 196 5). These bone m arrow-derived lymphocytes w ere
shown to
be
present in large numbers in the spleen (Tyan and Cole,
19 65) , lymph nodes ( Billingham an d Silvers, 196 4), an d thym us ( Sosin
e t
al.,
1966; Stutman a nd Good, 19 69 ). In t he rat, i t has been shown th at
the cells responsible for GVHR are bone marrow-derived but mature
elsewhere ( McGregor, 19 68). Thoracic du ct cells were obtained from F,
hybrid rats (interm ediate hos t) which had been inoculated at birth with
parental strain bone marrow cells. The thoracic duct cells were then
transferred into
a
second F, hybrid recipient of
a
different genotype.
A
GVHR was regularly observed in these latter recipients. However, the
capacity of the thoracic duct cells of the F, intermediate host to transfer
GVHR was diminished if spleen cells and not bone marrow cells were
initially injected into the intermediate host. Thoracic duct cells from
normal uninoculated
F,
hybrids failed to give the reaction in the re-
cipients. However, the bone marrow is not the source of mature GVHR
cells since it is less potent than the thoracic duct cells in transferring
GVHR to
F,
hybrids. Furthermore, thoracic duct cells from rats injected
neonatally with bone marrow cells obtained from lyniphocyte-depleted
adult donors were as effective as thoracic duct cells obtained from rats
injected neonatally with bone marrow cells obtained from norma1 un-
treated donors in transferring the GVHR to other recipients ( McGregor,
19 68 ). This would indicate that i t is the bone marrow cell and n ot th e
circulating small lymphocyte o r th e lymphocyte residing in th e peripheral
lymphoid tissues which can best transfer GVHR in the rat. In the mouse,
Tyan and Cole (1965) were able to show that a significant number of
deaths occurred among F, hybrid hosts when they had received spleen
cells from parental mice which had been injected with chromosomally
marked, adult, bone marrow cells. Spleen cells from mice that had not
been injected with the marrow or had been injected with thymus cells
only were less effective.
C.
THE
DEL AYE D YPERSENSITIVITYEACI-ION
The successful induction of the delayed hypersensitivity reaction
appears to requ ire the interaction of two cell type s in th e rat-a thymic-
derived cell for the initial sensitization step(s) and a bone marrow-
derived cell which participates in the subsequent cellular infiltrative
reaction (Lubaroff and Waksman, 1967, 1968a,b). The involvement of
th e thymus o r of th e thymus-derived cells in th e delayed hypersensitivity
reaction is illustrated by demonstrating the effects of neonatal thymec-
tomy. In the rat, neonatal thymectomy prior to sensitization with the
antigen inhibits the subsequent delayed hypersensitivity reaction. On the
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250
NABIH I. ABDOU AND MAXWELL RICHTER
other hand, thymectomy of adult Lewis rats followed by sensitization
with tubercle bacilli does not inhibit their ability to develop delayed
skin reactions (Jankovic
e t
al.,
1962; Miller
et
al.,
1962). Neonatally
thymectomized animals can become sensitized if they are injected with
sensitized syngeneic lymphoid cells prior to challenge. These data
indicate that the thymus is required for the active induction of cellular
immunity but not for expression of the infiltrative reaction.
The existing evidence strongly suggests that the majority of cells
infiltrating the site of the delayed hypersensitivity reaction are not
actively sensitized cells, but rather are circulating cells which tend to
accumulate, in a random fashion, at the site of the lesion (McClusky
et
al.,
1963; Turk and Oort, 1963;
S.
Cohen
e t
al.,
1967; Najarian and
Feldman, 1963a,b). In all of these studies, sensitized radioactively labeled
lymphoid cells were transferred to normal recipients or recipients sensi-
tized with a different antigen, in whom lesions accompanied by cell
infiltrates were induced by the injection of the specific (antigen used
to sensitize the cell donor) and nonspecific ( recipient-specific) antigen.
On the basis of radioautographs of biopsy sections or radioactive analyses
of tissue specimens, it is generally agreed that the majority of the mono-
nuclear cells infiltrating the specifically sensitized sites are “‘nonsensitized
cells. These cells are phagocytic and resemble macrophages that appear
in sites of nonspecific inflammation (Volkman and Gowans,
1965).
The
experiments of Lubaroff and Waksman (1968a,b) demonstrate that the
successful transfer of tuberculin hypersensitivity with sensitized lymph
node cells to thymectomized irradiated recipients depends on the simul-
taneous or prior injection of normal bone marrow cells. Normal thymus,
spleen, lymph node, or peritoneal exudate cells, even at high doses, could
not be substituted for the bone marrow in producing the tuberculin
reaction. These experiments indicate that once sensitization has occurred,
a bone marrow cell and not a thymic cell is required for the manifesta-
tion of the delayed hypersensitivity reaction. The precise origin
of
the
cells infiltrating the skin was investigated by the administration of allo-
geneic bone marrow to the thymectomized irradiated rats prior to the
administration of the sensitized lymph node cells. Fluorescein-conjugated
antiserum against the cells of the bone marrow donor and recipient was
then applied to the biopsies
of
the sites of skin reaction. The majority of
the cells were shown to be derived from the infused (donor) marrow.
The relative percentages of marrow-derived and lymph node-derived
cells in the tuberculin reaction remained the same during the 9-24-hour
period following the skin test (Lubaroff and Waksman, 1968b).
Histological analyses
(
Spector, 1967) and studies with labeled cells
suggest that a simiIar mechanism is involved in other reactions similar to
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251
OLE
OF BONE MARROW IN
THE
IMMUNE RESPONSE
the tuberculin-induced reaction, such as autoallergic lesions (Kosunen
e t al., 19 63), the skin homograft reactions (Pren derg ast, 196 4), dis-
seminated lesions of adjuvant arthritis (Bu rstein an d Waksman, 19 64 ),
and contact allergy (McClusky et nl., 1963). In the latter, Liden (1967)
has shown that bone marrow cells contribute to the formation of the
mononuclear infiltrate a t skin sites of allergic contact d erm atitis induce d
by dinitrochlorobenzene in the guinea pig. Local in situ labelling of the
bone marrow cells of the experimental animal with tritiated thymidine
results in their emigration from the bone marrow and their accumulation
in the skin lesion. The labeled cells were also detected in the regional
lymp h nodes an d in nodes at sites unrelated to the area of sensitization
(L id en and L inna, 19 69) , thus indicating th e nonspecific nature of the
bone marrow participation in this reaction. The demonstration of par-
ticipation of two cell types in contact allergy
was
confirmed morphologi-
cally b y Davies et al. (19 69 ). They described a b iphasic response in the
regional nodes of mice painted on the skin with oxazolone. An initial
paracortical proliferative response of thymus-derived cells was followed
by medullary hyperplasia and with germinal center formation, composed
mainly of bone marrow-derived cells.
VIII.
Cells
Affected
in
Immu n o l o g i c a l Tolerance
Immune tolerance is considered to be due to a depletion of immuno-
competent cells specifically reactive to the tolerogenic antigen or to an
altered reactivity of these cells so that they can no longer recognize the
antigen (rev iew ed in Dresser an d Mitchison, 19 68). T he failure to respond
with antibody formation could be due to either interference with the
access of antigen to the reactive cells (afferent limb of the immune
response) or interference with the synthesis and release of antibody by
the AFC (efferent l imb of the immune response). Evidence for failure
at the cellular level of the immune response has been shown by several
investigators (Billingham et al., 1956; W eigle an d Dixon, 1959; Friedm an,
1962; Battisto and Chase, 1963; Sercarz and Coons, 196 3b) . It has be en
demonstrated that the immune response can be induced in the immuno-
incompetent host by the transfer of cells of normal lymphoid tissues
(lymph node, thymus, or spleen cells) (Brooke and Karnovsky,
1961)
an d tha t lymphoid tissues from t he tolerant donors fail to give a response
in irradiated recipients (Mitchison, 1963; Martinez and Good, 1963;
McGregor e t
al.,
1967), thus demonstrating clearly that the immuno-
incompetent cell in the paralyzed animal is the smalI lymphocyte.
McCulloch and Gowans (1967) have shown that populations of small
lymphocytes from the thoracic duct
of
rats m ay sho w full immune re-
activity or partial or complete tolerance toward either histocompatibility
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252
N A B M I. ABDOU AND MAXWELL RICHTER
antigens or sheep erythrocytes, depending on the immune status of the
cell donor with respect to these antigens. Since thoracic duct lymphocytes
contain both ARC and AFC, it cannot be concluded from this study
which type of cell is tolerant in the immune tolerant state. However,
recent studies, using
Salmonella adelaide
flagellar antigen ( Armstrong
et d. 1969), SRBC (R.
K.
Gershon et al.,
1968;
Many and Schwartz,
1969), BSA (Taylor, 1968)) BGG (Isakovic
e t
al., 1965; Staples
et
al.,
1966), and tumor antigens (Abdou and McKenna, 1968) have shown
that the thymic ARC is the site of the lesion in the immune tolerant state
in the mouse and rat. This conclusion is based
on
the fact that the thymus
in these animals is the source of the ARC and in the failure of the trans-
ferred “tolerant” thymus cells to mediate an immune response in an
immunoincompetent irradiated or neonatal host with respect to the
tolerogenic antigen although the response to other antigens is normal.
However, it may be that the tolerant donor thymus is not tolerant due
to any effect on the thymic ARC; rather the tolerance may be attributed
to the ARC having vacated the thymus following interaction with the
antigen, much in the same way as the rabbit bone marrow ARC has
been considered to vacate the bone marrow
8-48
hours following inter-
action with the antigen in viuo (Abdou and Richter, 1969a,c).
Recently, however, Playfair (1969) has shown that bone marrow cells
obtained from adult mice made tolerant to SRBC through the combined
injection of SRBC and cyclophosphamide were unable to transfer im-
munocompetence to irradiated recipients, even when transferred along
with normal thymic cells. This finding suggests that the ARC is not the
cell affected in drug-induced tolerance or that its organ of habitation
may be altered as
a
result of the administration of the cyclophosphamide.
Abdou and Richter (1969~) ave demonstrated that rabbits made
tolerant at birth to HSA or BGG could be rendered immunocompetent
with respect to the tolerogenic antigen by the administration of normal
bone marrow ARC. These results demonstrate that the AFC is unaffected
in the tolerant animal. Moreover, bone marrow cells taken from tolerant
rabbits failed to transfer immunocompetence to irradiated immuno-
incompetent rabbits, in whom the ARC had been inactivated by the
irradiation but which still possessed the normal complement of AFC
(Richter and Abdou, 1969). Since the AFC is still capable of responding
to the tolerogenic antigen, provided normal allogeneic ARC are injected
into the animal, it would appear that the lesion in the immune tolerant
state is at the level of the ARC (Fig. 4).Whether the ARC are, in fact,
still present in the host in an inactive state or whether they no longer
exist cannot be ascertained from existing data. However, our data
(Abdou and Richter, 1969c) permit for an understanding for the failure
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ROLE
OF
BONE
MARROW
IN THE IMMUNE RESPONSE
253
IMMUNE R E S P O N S E
A9
Ab
IMMUNOLOGIC TOLERANCE
TOLERANT
Ag
1
RC ARC
A FC
Ab
FIG.4.
Cellular interaction5 resulting in the induction of the immune response
and immunological tolerance.
(
ARC-antigen-reactive cells; AFC-antibody-fonning
cells; Ag-antigen; Ab-antibody. )
of Sercarz an d Coons (1 96 3a ,b) to dete ct A FC in the tolerant animal.
Although th e AF C is capable of responding to th e antigen, i t never gets
the “message” since the block occurs at a stage prior to that of AFC
function (Fig.
4) .
That the macrophage is not the tolerant cell has been shown by the
studies of M itchison (19 69 ) an d
G.
Harris (19 67 ) who observed that
macrophages obtained from peritoneal exudates of tolerant animals can
take up the tolerogenic antigen and can initiate a specific immune
response in both tolerant and normal recipients. Macrophages from para-
lyzed mice were as active as normal macrophages in generating PFC
in vitro when allowed to interact with suspensions of lymphocytes ob-
tained from spleens of normal syngeneic mice ( Fo rb es , 19 69 ).
Self-recognition can be altered by contact of bone marrow cells with
allogeneic red cells ( Uphoff, 1 96 9a ,b). Lethally irradiated mice failed
to accept syngeneic skin grafts if given syngeneic bone marrow that had
been incubated in
vitro
with allogeneic erythrocytes. Moreover, the
in vitro
incubated bone marrow could protect the allogeneic but not the
syngeneic host against the lethal effect of irradiation and was unable
to indu ce secondary disease in th e allogeneic host (t h e red cell donor)-
a disease induced in 100% of irradiated mice injected with allogeneic
bone marrow cells not incu bated
in
vitro with erythrocytes. These results
suggest tha t , during th e
in
vitro
incubation of th e allogeneic erythrocytes
with the bone marrow cells, the latter are modified in such a way that
they no longer recognize the allogeneic strain as foreign nor the syn-
geiicic strain as self. However, in view
of
the unique nature of these
findings and the absence of confirmatory findings using this and other
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254
N A B I H
I.
ABDOU AND
MAXWELL RICHTER
antigens, the significance and interpretations of these findings may be
presumptuous at this time.
The mechanism whereby immunological tolerance is induced must
surely be related to the fact that the ARC and the AFC are two separate
cell entities. This “division of labor” permits for the exclusion of ARC
of a particular specificity without affecting the general immune respon-
siveness of the host. On the assumption that ARC exist early in fetal life,
their interaction with autologous antigens to which they may be com-
mitted (“forbidden clones”) may result in death of this cell population,
much in the same way that fetal exposure to antiallotype serum results
in subsequent failure of the immunologically mature adult rabbit to syn-
thesize immunoglobulin
of
this particular allotype
(
Marcuson and Roitt,
1969, Mage and Dray, 1965; Lummus et al., 1967 ). The ease with which
immunological tolerance may be accomplished in prenatal and neonatal
life, as compared with the difficulty encountered in attempting to induce
tolerance in adult animals ( Smith, 1961; Dresser and Mitchison, 1968),
may be attributed to the absence of scarcity of functioning macrophages
in the fetus and neonate. It has been demonstrated that the transfer of
peritoneal exudate cells into isogeneic neonatal recipient mice results in
a markedly enhanced immune responsive state, thus suggesting that
the immunoincompetence in the neonatal state may be attributed more
to a deficiency of macrophages than to an immature lymphoid cell
system
(
Argyris, 1969a). It would, therefore, not be too presumptuous
to assume that the fetus and neonate are devoid of functional macro-
phages, the function
of
which is considered to be that
of
antigen trapping
and processing, resulting in the release of highly immunogenic forms
of the original antigen. It is considered that interaction
of
the ARC with
the native antigen results in the induction
of
a tolerant state in this
cell, whereas interaction of the ARC with the macrophage-processed
form of the antigen sets
off
the intra- and intercellular reactions culminat-
ing in antibody formation (Richter, 1970). In the absence of a func-
tional macrophage population of cells, as occurs in the fetus and neonate,
the native antigen can react directly with the ARC, thus rendering it
permanently incompetent or resulting in cell death. In the immunologi-
cally mature animal, on the other hand, the presence
of
the macrophages
would tend to “protect” the immunocompetent ARC from the native
antigen (Nossal
e t
al., 1966; McKhann, 1969).
Recent findings using nonphagocytizable antigens support the above
interpretation as to the role of the macrophage. Aggregate-free prepara-
tions
of
HGG or
BSA,
obtained by ultracentrifugation or gel filtration
of
the “native” antigen or filtration through the reticuloendothelial system
of an intermediate animal (Biro and Garcia,
1965;
Frei et al.,
1965, 1968),
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ROLE OF BONE
MARROW I N
THE
MMUNE RESPONSE
255
induce
a
state of tolerance rather than immunity following their injec-
tion into adult recipients. The induction of tolerance in the adult animal
is probably facilitated by the bypass
of
the macrophage, resulting in
direct interaction of the native antigen with the ARC (Abdou and
Richter, 1970b).
IX. Bone M a r r o w Transp lanta t ion-App l ica t ion
The transplantation of bone marrow for the treatment of various
blood dyscrasias in animals was first attempted in the late nineteenth
century (re fer red to in Murphy, 19 14 ). Th e benefits derived from it
were rather equivocal. The concept of heinatopoietic tissue transplanta-
tion
as
a postirradiation the rap eu tic measu re has, however, end ure d.
Shielding of the exteriorized spleen or implantation of hematopoietic
tissue (infant spleen) in mice subjected to lethal irradiation results in
enhanced survival (Jacobson et al., 1951 . Rekers and co-workers ( 1950)
attempted marrow transplantation in irradiated dogs and obtained
slightly favorable results as reflected by small differences in mortality and
hematological responses between experimental and control animals.
Lorenz and co-workers (1952) demonstrated the protective effects of the
postirradiation injection of syngeneic bone marrow in 70 to 95% of
lethally irradiated mice and guinea pigs, They subsequently extended
their studies to show that allogeneic and xenogeneic bone marrow trans-
plants in irradiated mice are less effective if compared to syngeneic
marrow transplants.
By using markers, it was established that the transplanted bone
marrow is accepted by the immunoincompetent host so that the re-
cipient’s hematopoietic cells became of donor type. Among the markers
used are chromosome markers in man (Bach
et
al., 1968; Gatti
e t al.,
1968) and mouse ( Ca r ter e t al., 19 55 ), differences in karyotype between
mouse and rat and between normal mice and those with a visibly ab-
normal chromosome (C.
E.
Ford
e t
al., 1956), blood group antigens
( Lindsley et al., 1955; Makinodan, 19 56 ), histocompatibility antigens
( Mitchison, 1 95 6), histochemical differences in alkaline phosp hatase
activity of mouse and rat granulocytes (Nowell et
al.,
1956) , and the
differences in hemoglobin concentration
of
progeny of stem cells of
donor and recipient origin (Popp e t al., 19 58 ). These investigations all
demonstrated that the donor bone marrow can populate and replace
the recipient’s blood elements.
These initial results provided an im petus for more extensive investiga-
tions involving bone marrow transplantation in animals and man for
correction of abnormal hematopoiesis or various immune deficiency syn-
dromes. Congenital macrocytic anemia of strain WWv mice could be
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256
NABIH I. ABDOU AND MAXWELL RICHTER
corrected by the administration of hematologically normal allogeneic
( D B A / H T,T,) hematopoietic cells, By studying the anemic mice for
red cell hemoglobin and bone marrow cells for the chromosome marker,
it was shown that the hematopoietic systcm of the anemic mice was
totally replaced by the donor stem cell (Seller and Polani, 1966, 1969).
Cases of aplastic anem ia in hu m an twins could be cu red by bone marrow
infusion from the twin donor (Ro bins an d Noyes, 1961; S. D. Mills et al.,
1964; Thom as et al., 1964). Bone marrow transplants have also been used
with variable degrees of success in radiation sickness, overdosage of
immuno suppressants (re vie we d in Lou tit, 19 65 ), an d in some cases of
leukemia (rev iew ed in Math6, 1960; Math6 e t al., 1965) in animal and
man. Successful treatment of leukemia and lymphoma by lethal irradia-
tion or massive doses of immunosuppressants followed by infusion of
syngeneic marrow cells in animals an d man h ave been repo rted (Atk. nson
et
al., 1959; Thomas
et al.,
1959; Floersheim, 19 69 ). Allogeneic bo ne
marrow transplants have proved to be unsuccessful (revie we d in M ath&,
1968).
In the combined humoral and cellular immune deficiency syndrome
in man ( lymphopenic or Swiss-type hypogammaglobulinemia), it has
been postulated th at t he defect is at the stem cell level. Since graft re-
jection is impaired in these patients, attempts at restoration of immune
competence by grafting closely matched bone marrow cells have been
successful (Gatti e t
al.,
1968; Meuw issen et al., 196913; de Koning et al.,
1969) . To avoid the risk of GVHR, special treatment was given to donor
bone marrow lymphocytes either by incubating the marrow cells at 37"
C. for 2 hours in th e presence of antilymp hoblast serum (Meu wissen et al.,
196913) or
by
fractioning the bon e marrow cells in albumin gradient and
infusing cells devoid of their small lymphocyte co ntent ( d e Koning et al.,
19 69 ). These types of treatm ents w ere shown to red uc e the num ber of
immunocompetent cells in the transferred marrow (Math6 et
al.,
1966;
Najarian et al., 1969; Dicke et al., 1968).A case of Wiscott-Aldrich syn-
drome has also been successfully treated with a bo ne m arrow transplant.
Evidence of a chimeric state was detected
a
few weeks following the
transplant ( Bach e t al., 1968) .
There are certain risks in transplanting bone marrow to immuno-
incompetent recipients. Graft-versus-host reaction is frequently seen
after
the
establishment of an allogeneic chimera due to interaction of
immunocompetent cells of the grafted marrow with the host tissues,
which contain transplantation antigens absent in the cell donor. The
GVHR is characterized by wasting, diarrhea, skin rash, fever, spleno-
megaly, and hepatomegaly (reviewed in Simonsen, 1962; Math6 e t al.,
1967; Kretschmer e t
al.,
1969; Hathaway et al., 1965; Uphoff and Law,
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258
NABIH I.
ABDOU AND MAXWELL RICHTER
The schemes and concepts presented should not be viewed as de-
picting the complete or ultimate picture since other types
of
cel l and/or
mediators may be involved.
We
have, instead, presented
a
concept which
is flexible, which stresses the dynamic rather than static nature of t he
various cell compartments participating in the immune response, and
which is amenable to verification in the laboratory.
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Cell Interaction
i n
Antibody Synthesis'
D.
W.
TALMAGE,
J .
RADOVICH, AND H. HEMMINGSEN
Depar tment o f Microb io logy, Un ive rs i fy of Colo rado Med ica l Cen te r , Denver, Colorado
I. Introduction . . . . . . . . . . . .
11.
Two
Universes of Immunocompetent Cells . . . . . .
111. The Adherent CelI
. . . . . . . . . . .
IV. Antigenic Com petition . . . . . . . . . .
V. Enhan cing Effe ct of Multiple Antigenic Determinants
. . . .
VI. En han cin g and Suppressive Effects of Passively Adm inistered Antibody
VII. Discussion and Speculations . . . . . . . . .
References
. . . . . . . . . . . . .
271
272
273
276
276
277
277
279
I.
In t roduc t ion
Immunologists have been prone to construct complex models of cell
interaction and cell differentiation to explain the observations that they
or their contemporaries have made on various features of the immune
response ( Wissler e t al., 1957; Ellis e t
al.,
1967; Makinodan and Albright,
1967; Sterzl, 1967; Makinodan
et
al.,
1969). The initial models were based
on histological descriptions of the localization of injected antigen and led
to
the concept of cooperation between a macrophage, which digested
and processed the antigen, and a cell, which responded to the modified
antigen by making antibody (Fishman, 1959, 1961; Fishman and Adler,
1963, 1967; AdIer e t
a?.,
1966; Feldman and Gallily, 1967; Gallily and
Feldman, 1967). This concept was supported by experiments in which
extracts of cells previously exposed to antigen were shown
to
be capable
of inducing antibody formation in normal lymphocytes (Cohen and
Parks,
1964).
In the last few years it has been possible to demonstrate with sepa-
rated cell populations that an interaction between two or more living
cells was required for the antibody response to at least some antigens
both
in
vivo (Claman
e t
al., 1966a,b, 1968; Claman and Chaperon, 1969;
Davies e t
al.,
1966, 1967; Davies, 1969; Miller e t al., 1967; Miller and
Mitchell, 1967a,b, 1968, 1969a,b; Miller and Osoba, 1967; Gershon e t al.,
1968; Mitchell and Miller, 1968a,b; Nossal
e t al.,
1968; Radovich
e t
al.,
1968; Talmage e t al., 1969a) and in
vitro
(Dutton and Mishell, 1967a,b;
Mishell and Dutton, 1967; Mosier, 1967, 1969; Mosier and Coppleson,
1968; Raidt
e t
al., 1968; Pierce and Benacerraf, 1969; Roseman, 1969;
'This work was supported
by
Grant No. 5 R01 AI03047-11 from the National
Institute of Allergy and Infections Disease.
271
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272
D. W. TALMAGE, J. RADOVICH, AND H. HEMMINGSEN
Talmage et nl., 1969b). These findings have made it necessary to re-
appraise many of th e widely accep ted an d basic concepts of imm unology,
such
as
th e clonal selection theory, th e role of th e macrop hage referred
to above, and the difference between cells responding to the first and
second injections of antigen.
In the present paper we shall first review the recent observations on
th e interactions of sepa rate popu lations of cells from inb red m ice an d
relate these to experiments with congenital dysfunction of the immune
system in humans and surgical removal of the thymus in mice and the
thymus and bursa in chickens, We shall the n briefly review thr ee related
phenomena: antigenic competition, the carrier effect of multiple anti-
genic determinants, and the suppressive and enhancing effects
of
pas-
sively administered antibodies. Finally, we shall attempt to discuss
briefly the more likely hypotheses which have survived.
II. T w o Universes of l m m u n o c o m p e t e n t Cells
Birds have
two
clearly defined organs for differentiation of immuno-
competent cells, the
bursa
of Fabricius and the thymus (Warner e t al.,
1962; Warner and Szenberg, 1962, 1964; Szenberg and Warner, 1964;
Warner, M 4 ) . Surgical removal of one or the other of these organs from
the newly hatched chick produces distinctly different deficiencies in im-
munological capacity. Loss of the bursa affects serum immunoglobulin
levels, formation of secondary nodules in spleen and lymph nodes, and
humoral antibody responses (Cooper et al., 1965, 1966; Ca in e t at., 1968,
1969; Meuw issen et al., 1969a,b; Van Meter et al., 1969). Early loss of
the thymus produces a deficiency in delayed hypersensitivity and impair-
ment of homograft rejection (Jankovich et al., 1962; Aspinall e t al., 1963;
Cooper
et
al., 1968; Good e t al., 1968) .
The conclusion that the role
of
the thymus concerns the differentia-
tion of cells involved in delayed hypersensitivity is borne out in humans
with congenital absence of the thymus
(
DiGeorge syndrome). These
individuals have complete failure of delayed hypersensitivity, and their
lymphocytes are not stimulated by phytohemagglutinin (August e t al.,
1968; DiGeorge, 1968; Meuwissen et at?., 1969a,b). However, they may
have normal immunoglobulin levels, secondary nodules in spleen and
lymph nodes, and make vigorous antibody responses to certain antigens.
On the other hand, individuals with sex-linked hypogammaglobulinemia
have a normal thym us a n d delayed hyp ersensitivity ( Bruton, 195 2). Thus
there appear to be two distinct universes of immunocompetent cells, one
derived from th e thymus an d th e other derived from the bu rsa of Fabri-
cius in birds. In mam mals th e source of th e second cell type is not estab-
lished.
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CELL
I N TER A C TI ON IN A N TI BOD Y S Y N THES I S
273
Although most immunologists would agree in general with the above
statements, controversy develops over evidcmce for interaction between
the two cell types. Neonatally thymectomized mice ( a n d probably
humans with DiGeorge syndroine ) do not have
a
normal hum oral response
to all antigens. The response to sheep red cells and heterologous serum
proteins is absent or reduced, whereas the response to bacterial and viral
antigens a pp ear s norm al (Miller, 1962; Miller an d O soba, 1967; Rothb erg
an d Rob ert, 1967; Neselof, 1968; Armstrong, 1969; Armstrong
et
al., 1969;
Davies, 1969; Miller a nd h4itchel1, 196 9a ,b ). This has led to the concept
tha t there a re thymus-dependent an d thymus-independent antigens. How -
ever, the separation
of
th e two types of a ntigen varies with species; in
the rat, the response to sheep red cells
is
t l iymus-independent (Claman
et nl., 1966a,b; Pinnas an d Fitch , 196 6) , whereas in th e mouse this antigen
is thymus dependent .
Direct evidence for interaction between the two universes of cells
was first fou nd by Clam an et nl. ( 19 66a J)). X-Irradiated mice repopulated
with thymus or bone marrow cells alone made very poor immune re-
sponses to sh eep red blood cells. W hen both cell types wer e injected th e
response was much higher than the sum of the two individual responses.
It
was soon shown that the bone marrow, not the thymus, contributed
the precursor of th e antibody-forming cell ( P cell) (Davies e t
aZ.,
1967;
Mitchell an d Miller, 1968a,b;Nossal et ol.,
1968;
Talmage
e t al.,
1969a) ;
however, th e question of th e role of the thynius cell in hum oral an tibody
production to the so-called “thymus-dependent antigens” was left un-
answered.
Ill. The Adherent Cel l
More recently, Mosier showed that an interaction between two kinds
of cells could be demonstrated
in
the in oitro response
of
mouse spleen
cells to shee p red blood cell antigen (Mo sier, 19 67 ). Th e spleen cells
could be sepirated into a population adherent to glass or plastic and
a nonadherent population. A synergism between the two populations
could be demonstrated.
The
adheren t cel l ( A cel l) does not appear to be the precursor cell
( P cel l) nor th e thymus cel l ( T cell) required for the
in
vivo response
in X-irradiated mice. Nonadherent spleen cells separated on a glass bead
column will give an excelleiit responsc when injected into X-irradiated
mice but are complctely unresponsive in v i f ro (Talniage et
al.,
1969a,b) .
The probable reason for this distinction was found by Roseman, who
demonstrated that the adherent cell
is
relatively resistant
to
X-radiation
(1969) in contrast to the marked sensitivity of the other two cells
(M osier, 1967; Mosier e f nl., 1969; Roseman, 1969; Roseman et al., 1969) .
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274 D. W. TALMAGE,
J.
RADOVICH, AND
H.
HEMMINGSEN
Thus the adherent cell is probably still functional in the X-irradiated
mouse for at least a short time after radiation. We have confirmed the
radioresistance of th e adh ere nt cell an d uscd this radioresistance to show
that there is a higher than normal concentration of adherent cells in the
spleens of X-irradiated mice reconstituted with bone marrow ( B M S C )
( T a b le I ) . If these mice were given a second dose of X-radiation
2
hours
before harvesting the bone marrow-derived spleen cells, there was little
or no loss of fu nction, b ut if t h e second dose of X-radiation w as given 24
hours befo re harvesting, loss of th e ad he re nt cell or its function was
marked. In these experiments spleens were taken from mice
7
days after
radiation (1000 r 6oCo)and the injection of syngeneic bone marrow. A
second dose of X-radiation ( 5 0 0 r ) was given to these animals
2
or
24
hours before sacrifice. T he cells from spleens ( B M S C ) taken 2 hours after
500r were far superior to X-rayed normal spleen cells in supporting the
in u i t ro response of a small number of immune spleen cells. However,
by
24 hours after X -radiation the effectiveness of the BMSC ha d decreased
markedly. This delayed effect of X-radiation on the adherent cell may
account for the delayed effect of X-radiation on the immune response
(Dixon et al.,
1952;
Pribnow and Silverman,
1967, 1969).
It thus appears that there are two different kinds of cell interactions.
TABLE I
THEFLAQ UE-FO RMI NGELLRESPONSEO SHEEPERYTH O CYTESn
vitro
U S I N G N T R E A TE DND X-IRRADIATEDO N EMARROW-RECONSTITUTED
SPLEENCELLS
N D
X-IRRADIATEDPLEEN ELLS
Norma'
Immune
BMSC.
BMSC' spleenb
cell ( X 10-6) cell ( X BMSCu**
500
1.) 500 r) 500 r )
Adherent spleen Untreated ( 2
hr.
after
(24
hr. after (2 hr. after
10
3
3944/6 3639/6
250/3
-
10 1 1414/9 1389/9 169/3 6/3
10 0 3 195/9 143/9 1312 2/3
10
0 1 38/12 5/12
10
20
2 - 1145/3
10 2
3850/3
4010/3 775/3
5
2 1.545~9 1530/3 -
2 . 5
2
243/3 670/3 -
-
-
11/3
1 3
-
a
BM SC-bone marrow (reconstituted ) spleen cells.
b
Average number
of
plaque-forming cells (PF C) number
of
cultures. Usually the
cells from tw o or three cultures were pooled and duplicate PFC assays performed on the
pool. In these experiments,
3 X 10-5
mm une spleen cell controls without added adherent
cells gave from
0
to 100
PFC.
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CELL
INTERACTION IN ANTIBODY SYNTHESIS
275
An interaction between
T
a nd P cells can be demonstrated by giving
thymus and marrow cells to the X-irradiated mouse. An interaction
between A ceIls and
a
mixture of
P
a nd
T
cells can
be
studied
in
vitro
(T a b l e I ) . Unfortunately, it has not been possible to separate the mixed
population of P and
T
cells in the spleen, and neither thymus nor bone
marrow cells work well in the in vitro culture system. Some evidence of
interaction within the population of immune spleen cells can
be
seen in
the data of Table
I.
T h e slope of th e cell dose-response curv e is con-
siderably greater than
1.
The function of the A cell is not clear. Possibly the antigen adheres
to it as to the dendritic cells of the lymph follicle. However, some im-
munologists have postulated that the antigen
is
ingested by macrophages
and “processed” in some way
to
make it functional (Garvey and Canip-
bell, 1957; Adler et al., 1966; Fcldman and Gallily, 1967; Fishman and
Adler, 1967; Gallily a nd Feldm an, 19 67 ). W e know of no convincing
evidence in favor of such a concept and there are two important bits of
evidence against it: ( I ) for several days after the injection of antigen,
the antibody response can be inhibited by an injection of specific anti-
body (Moller and Wigzell, 1965; Wigzell, 1966; Dixon
et
al., 1967;
Britton and Moller, 1968) (this suggests that the functioning antigen is
outside of cells); an d ( 2 ) the major part of the antibody response is to
antigenic determinants that are lost if the quaternary structure of the
antigen is broken down (Sela et nl., 1967; Henney an d Ishizaka, 19 68) .
TABLE I1
RESPONSEO SHEEP RED BLOOD ELLSO F m C ~ - I ~ ~ ~ ~ ~ECIPIENTS
RECEIVINGPLEENCELLS
A N D
HORSE
N D
SHEEPRED BLOODCELLS
A T V.4RIoUS INTERVALS AFTER
CELL TRANSFER”’)
10 x
106
Cells
50 x 106 Cells
transferred transferred
1Iice Response Mice Response
Groupr (No.) (I’FCld (No.) (PFC)d
I . 8-RBC
day
0
8 66 It 14
7
218 f
25
11.
S-RBC
day 4
7 181 f 9 8 102 f 7
111.
H-RBC
day 0;
S R B C
day 4
11
58
zk
7 12
12 f
i
a Five irradiated recipients given sheep red
blood
cells on day
4
h i t
no
spleen cells
averaged 8 plaqrie-forming cells (PFCI spleen.
b
From Rxdovich
and
Talmage
(1967).
d Mean
PFC
for S-RBC per inillioil cells tr;tnsferred in spleen of recipient 6 days after
S-RBC--sheep red
blood
cells; 11-I1BC-horse red blood cells.
iiijecl ion
of
8-RBC S.E.
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276
D.
W. TALMAGE, J . RADOVICH, AND H. HEMMINGSEN
IV.
A n t i g e n i c C o m p e t i t i o n
The relationship between the phenomenon of antigenic competition
and cell interaction can be seen in the data presented in Table 11. Here
th e depressive effect of one red cell antigen (h or se ) on th e response to
a second, apparently unrelated, red cell (sheep) is studied in the X-ir-
rad iate d mouse reconstituted with 10 or 50 million norm al spleen cells.
T he response with 50 million spleen cells is more than 5 times higher than
with 10 million, which is probab ly du e to th e greater ch ance of cell
interaction when cells are more concentrated. In any case, the result of
the greater response to the first antigen in the 50-million group is a
greater depressive effect on the response to the second antigen.
Initially, the experiment of Table I1 was interpreted
as
suggesting
the existence of a suppressive humoral factor which was produced by
the response to the first antigen (Radovich and Talmage, 1967). [The
production of such
a
factor has been implicated in quite different experi-
ments by Ambrose (19 69 ).] How ever, the demonstration of an inter-
action between several cells requires a reassessment of this conclusion.
At least one of the cells involved in the immune response must be non-
specific, an d this cell may b e used up in th e response to the first antigen.
This
preemption is more likely to take place with
a
high concentration
of
interacting cells than with a low concentration.
V. Enhanc ing Ef fec t of M u l t i p l e A n t i g e n i c D et e r m i n an t s
The importance of a carrier molecule to the immune response of
simple chemical determinants (haptens) has been well known since the
experiments of Landsteiner ( Landsteiner and Lampl, 1917; Landsteiner,
1919, 1921, 1962). More recently it has been shown that the antigenic
determinants on the carrier molecule are important in the response to
the hapten. Protein molecules to which the injected animal has been
made tolerant are poor carriers, whereas the response to the hapten is
enhanced if the animal has been preimmunized to the carrier. Apparently,
the haptenic determinant occupies a position on the carrier molecule
equivalent to native determinants. If an animal is preimmunized to a
haptenic determinant complexed with one carrier, this will increase the
antibody response to native determinants of a completely different
carrier if the latter is complexed to the same hapten; and the addition
of two haptenic determinants is better than one in inducing an antibody
response to the carrier (Haurowitz, 1936; Cinader and Dubert, 1955;
Dixon a nd M aurer, 1955; D ub ert, 1956; C ina de r and Pierce, 1958; Salvin
and Smith, 1960; Weigle, 1962, 1964, 1865a,b; Ashley and Ovary, 1965;
Linscott and Weigle,
1965;
Dietrich, 1966; Green
et
d. 966; Leskowitz
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CELL INTERACTION IN ANTIBODY SYNTHESIS
277
et al., 1966; Yoshinmra an d Cinad er, 1966; C ina de r e t al., 1967; Fronstein
et
al., 1967; Levine, 1967; St. Rose and Cinader, 1967; Maurer and Pin-
chuck, 1968; Plescia
et
al.,
1968; Rittenberg and Campbell, 1968; Landy
and Braun, 1969; Leskowitz, 1968; Plescia, 1969; Rajewski, 1969).
The above findings are well explained by the requirement for
cell
interaction in the antibody response. If the antigen is an impo rtant factor
in cell interaction, such interaction will be greatly enhanced by the
presence of many determinants on the antigen molecule.
If
this concept
is correct, then the greater enhancing eff ect of two antigenic determinan ts
com pared t o one determinan t suggests that at least on e of the cells involved
is antigen-specific. It also suggests that cells specific for different deter-
minants may interact and enhance each other's response.
VI.
Enhanc ing and Suppress ive Ef fec ts of
Pass ive l y Adm in i s te red An t i body
It is well establishcd that passively administered antibody can both
suppress and enhance the active response to an injected antigen (Jerne,
1967; Pearlman , 1967; Ce rottini et
aZ.,
196 9). In general, the demonstra-
tion of an enhancing effect is demonstrable only with small doses of
antigen which produce less than a maximal response. Although the sup-
pressive effect of antibody is limited to the specific determinants repre-
sented in the antibody population, the enhancing effect of antibody is
nonspecific and applies to other determinants on the antigen molecule
(Pearlman, 1967).
If
the antigen is important in achieving cell interaction, then antibody
in excess, by blocking the antigenic determinants, will inhibit such inter-
action. An effect of antibody on blocking the aggregation of antibody-
forming cells has been demonstrated in vitro by Mosier (19 69) . Th e
prevention
of
clumping by this means or by mechanically reducing cell
movement (Mishell and Dutton, 1966, 1967) was shown to inhibit anti-
body formation.
T he nonspecific enh ancing effect of antibody suggests th at a t least one
of the cells involved in the interaction can
fix
antibody to its surface.
T h e presence of th e antibody will thus a id
in
the fixation of antigen and
other cells coated with antibody. Lan g and Ada (19 67 ) showed that the
presence of a small am oun t of antibo dy enha nces th e fixation of an tig en
to the dendritic cells of the lymph follicle.
VII.
Discussion
and
Speculat ions
The finding that there is a rcquirement for cell interaction in the
antibody response to some antigens has greatly complicated the problem
of interpretation of much experimental data. A major problem is the
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278
D. W.
TALMAGE,
J .
RADOVICH, A N D
H.
HEMMINGSEN
specificity of cell potential. The observation that single cells make only
one antibody (Green
e t al.,
1967; Makela, 1967) and that cells respond-
ing to one antigen may be selectively destroyed (Dutton and Mishell,
1967a) probably indicates that at least one of the interacting cells is
specific. O ne possibility is tha t th e two se pa ra te universes of cells, thymic
and extrathymic, are both highly specific. However, in such a case there
must be cells of each universe specific for different antigens and the
chance of two such cells meeting and interacting is very small if they
must both be specific for the same antigen.
A related question is the nature of the cell interaction and the sub-
stance th at passes from th e auxilliary cell
(T cell)
to the antibody-form-
ing cell
( P
ce ll) . If th e antibody-forming precursor is highly specific, th e
auxiliary cell probably provides only a nonspecific stimulus. However,
if
the
antibody-forming precursor is nonspecific, then it seems more
likely that some kind of specific informational ribonucleic acid or inducer
is transferred (Talmage et
al.,
1969a) .
One approach to this question has been to determine which cell is
the carrier of immune memory, since this memory is presumed to result
from th e selective change in nu m be r of specific cells, eithe r by selective
growth [positive memory or anamnestic responsiveness] or by selective
destruction [negative memory or tolerance]. Conflicting results have bee n
obtained in experiments designed to determine the locus of immunologi-
cal tolerance (Ta ylor, 1968; La ndy and Braun, 1969; Miller an d Mitchell ,
1969a; Playfair, 1 96 9) , an d it has n ot been clear whether the unrespon-
siveness involved w as cellular (c lo ne loss) or humoral ( d u e to feedback
immunosuppression).
It
has not been possible to study the locus of
enhanced responsiveness since no reliable method exists for separating
the two types of nonadherent cells after they have interacted.
What is the significance of thymus-independent antigens and the
immune responses in neonatally thymectomized mice and in athymic
humans? These responses appear to involve memory and, thus, cells with
specific potential (R oth be rg an d Ro bert, 1967; August et
al.,
1968;
DiGeorge, 1968; Kretshmer et al., 1968, 1969; Neselof, 1968; Rosen,
1968).
It is also apparent that if cell interaction is required in these responses,
one of the interacting cells need not be a thymus cell.
If extrathymic cells are able to interact and give immune responses,
then the simplest explanation for the fact that they are unable to do so
with some antigens is that t he extrathymic tissue contains too few of th e
right type of specific cells to ha ve a reasonabIe chan ce of ceIl interaction.
If this is the case, then thymus cclls will increase the chance of inter-
action only if they arc less specific than extrathymic cells. There is some
suggestion of this in experiments which indicate that less than 100
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CELL
I N T E R A C T I O N I N
ANTIBODY
S Y N T H E S I S
279
homologous lymphoid cells are needed to produce antigen-dependent
foci
on
the chorioallantoic membrane or graft-versus-host reaction in
chick embryos (Szenberg et
al.,
1962; Simons and Fowler, 1966; Simon-
sen, 1967).
If the above line of reasoning is correct, then the two separate uni-
verses of immunocompetent cells which have evolved have different
functions and different degrees of specificity. One is highly specific and
highly specialized for humoral antibody production and the other is
several orders less specific and adapted to cell-cell interactions. The
function
of
the thymus would be to decrease the specificity or rather
to increase the range of responsiveness of immunocompetent cells. In the
process the thymic cell loses its ability to export antibody to the serum
but gains a much greater ability to interact with other cells.
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The Role of Lysosomes in Immune Responses’
GERALD WEISSMANN’ AND
PETER
DUKOR
De p o r tme n t of Medic ine (Ce l l
B i o l ogy ond
Genetics) , Ne w
Y o r k
University
School o f Medic ine , New Y o r k , N e w York
I. Introduction . . . . . . . . . .
A. Abbreviations
. . . . . . . . .
11. Processing
of
Antigen by the Vacuolar System
. . .
A. A
Role for Macrophages in the Afferent Limb of the
Immune Response
. . . . . . . .
B .
The Localization of Antigen in Lymphoid Tissues .
C. The Fate of Antigen in Macrophages . . . .
111.
Mediators of Tissue Injury Found in Lysosomes
. .
IV. Lysosomes in Four Types of Iiiiiiiune Injury . . .
A. Type I Reactions . . . . . . . .
B.
Type
I1
Reactions
. . . . . . . .
C.
Type 111 Reactions . . . . . . . .
D.
Type IV Reactions . . . . . . . .
References . . . . . . . . . .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. .
. 283
. 284
. 285
.
285
.
288
. 290
. 304
.
306
.
306
.
309
.
311
. 316
. 322
I. Introduction
Described
as
a new class of subcellular organelles by de Duve et al.
(1955), ysosomes have been at the center of many studies in tissue
injury, inflammation, and immunity. Yet, as more data become available,
it is clear that the bulk of the lysosomal concept was foreshadowed by
th e works of E lie Metchnikoff (1905). His work overlaps the borders
between classical “humoral” immunology ( Ehrlich
)
and cel lular pa-
thology (Aschoff
).
In fo rmu lating the principle of phagocytosis, h e recog-
nized that uptake of foreign material was a primary event, common to
both immune recognition and acute inflammation. With deeper under-
standing of the role played by the “macrophages” and “microphages”
( polymorphonuclear leukocytes a nd lymphocytes ) in immunity, the
extent of Metchnikoffs insight becomes even more impressive. Although
he supposed wrongly that the complement of Bordet and Ehrlich was
elaborated by microphages, he deduced correctly that phagocytosis was
a necessary step for immune reaction to particulate antigens and that
uptake
of
particles per se could initiate sterile inflammation. It is toward
the recent ela bora tion of these concepts tha t this review will
be
addressed.
‘The original work described in this review has been aided by grants from the
’Career Research Scientist
of
the Health Research Council of New York (1-467).
National Institutes of Health (AM 08363 and AM 11949).
283
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284
GERALD WEISSMANN A N D PETER DUKOR
Lysosomes can be considered parts of a vacuolar system or, more
exactly, of an “exoplasmic reticulum” concerned with the digestion of
heterologous and autologous material (reviewed in
de
D uve a nd Wa t -
tiaux, 1966; Weissniann, 1967; d e D uv e 19 69 ). T h e organelles have also
been studied in those cells central to immune responses: macrophages,
monocytes, histiocytes, thymus cells, neutrophiles, eosinophiles, lympho-
cytes, etc. (s ee specific chapters in Dingle a nd Fell, 1969). Bu t we d o
not plan to discuss lysosomes by cell type; instead, we have divided
our subject into two major categories: 1 ) he processing
of
antigen by
th e vacuolar system-a responsibility chiefly of macrophages-and (2)
the mediation by lysosomes of immune injury-a responsibility chiefly
of microphages. These topics follow
as
naturally from the outlines
of
Metchnikoffs thought as they express themselves in terms coined by
de D uve.
A. ABBREVIATIONS
A list of abbreviations used in this chapter follows:
BAA
=
benzoyl-L-arginine amide
BCG = Bacillus Calmette-Gukrin
BSA = bovine serum albumin
cyclic AMP = cyclic adenosine 3’,5’-monophosphate
DNA = deoxyribonucleic ac id
EACA = 6-aminocaproic acid
L-GAT
=
poly- ( L-glutamic acid, L-alanine, L-tyrosine )
HSA = human serum albumin
L D H = lactic dehydrogenase
P H A = phytohemagglutinin
P P D
=
purified protein derivative
PP-L = protein polysaccharide (lig ht fraction of bovine nasal
P W M = pokeweed mitogen
RNA
=
ribonucleic acid
SRS-A =
slow reacting substance of anaphylaxis
TAME = tosyl-L-arginine methyl ester
L-TG = poly-( L-tyrosine, L-glutamic a c id )
D-TGA
=
poly- (D-tyrosine, D-glutamic acid , D -alanine)
(
T,G )-A-L
=
poly- (L-tyrosine, L-glutamic acid )-poly-m-alanine-
T L CK
=
tosyl-lysine chloromethylketone
T P CK = tosyl-phenylalanine chloromethylketone
cartilage )
poly-L-lysine
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THE ROLE
O F
LYSOSOMES
I N
IMMUNE RESPONSES
285
I I
Processing of A n t i g e n by t he
Vacuolar
System
A.
A
HOLEFOR MACROPHAGES
N THE AFFERENT
LIMB
OF
THE IMMUNE
ESPONSE
The induction of primary iminunc responses against many types of
antigens has been widcly recognized to require th e sequ ential inter-
action of foreign materials with two, or possibly three, distinct cell popu-
lations. Each of the properties
of
antigen-handling (Fishman, 1969),
of
antigen recognition, and of antibody synthesis (J .
F.
A. P. Miller and
Mitchell, 1969; A . J.
S.
D w ies , 1969; Clainan a nd C haperon, 1969; Taylor,
1969)
has
been attributed to an anatomically distinct cell line.
Since Metchnikoff (1S99, 1905) it has been appreciated that macro-
phages in th e liver, spleen, lymph nodes, an d peritoneal cavity can ingest
particulate and soluble materials and that these cells participate, at
least in par t, in the developm ent of imm unity. Antigen processing b egins
with endocytosis, which is a general phenomenon that occurs in the
majority of living cells (Jacques, 1969). Little evidence is available,
however, for implication
of
endocytic cells other than macrophages in
the induction of the immune response. It is this cell type which will
thereforc engage our interest. Interrelationships between macrophages,
the reticuloendothelia1 systtm, and immunity, have been the subject of
frequent and extensive reviews (Rowley, 1962; Thorbecke and Bena-
cerraf, 1962; Campbell and Garvey, 1963; Suter and Raniseier, 1964;
Mackaness an d Blanden, 1967; Nelson, 1 96 9) . T he re is general agree-
ment that mature macrophages do not themselves synthesize specific
antibodies ( Ehrich et
d.,
946; W eiler an d W eiler, 1965)
;
although possi-
ble exceptions to this rule will
be
dealt with below. It is likely that
macrophages play a more subtle and, probably, indirect role in the
afferent limb of th e immu ne rcsponse. T h e lines of evidence th at im-
plicate these cells in th e inductive stage of imm unity can be summarized
as follows:
In a variety of experimental models, the presence of macrophages
is a prerequisite for the induction of antibody formation. Although
lymphocytes from adult donors failed to confer immunological com-
petence to newborn rabbits (Dix on and Weigle, 195 7), peritoneal macro-
phages rendered such animals responsive
to
a protein antigen that
otherwise induced imniunological tolerance
(
Martin, 19 66 ). Similarly,
development of immunological reactivity to heterologous red cells was
accelerated in baby mice receiving macrophages from mature donors
(Braun and Lasky, 1967; Argyris, 19 68 ). Similar results w erc obtained
1.
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286
GERALD
WEISSMANN
AND
PETER
DUKOR
in attempts to restore immunological reactivity to bovine y-globulin or
Shigellu paradysenteriae
early after X-irradiation
of
rabbits ( Pribnow
and Silverman, 1967) or mice (Gallily and Feldman, 1967), respectively.
In both cases, injection of antigen with lymph node cells failed to elicit
antibody formation. However, immune responses were promptly induced
if lymph node cells were transferred together with macrophages which
had been incubated with the appropriate antigen
in vitro.
That pure
lymphocyte suspensions could be instructed by primed macrophages,
but not by antigen alone, was very elegantly demonstrated in experi-
ments of Ford
et
al. (1966). Rat thoracic duct lymphocytes were tempo-
rarily incubated with macrophages containing sheep red blood cells and
then freed of both phagocytes and antigen. Subsequently, only lympho-
cytes which had been exposed to macrophages were able to confer the
capacity to synthesize specific antibody to irradiated, syngeneic recipients.
The initiation of hemolysin formation by spleen cell suspensions
in
vitro
has been shown to depend on the presence of a macrophage-rich,
surface-adherent, and relatively radio-resistant subpopulation of cells.
This population fails to make antibody by itself, but upon incubation
with antigen it can stimulate antibody production by nonadherent cells
(Mosier, 1967; Mosier and Coppleson, 1968; Pierce, 1969; Roseman,
1969; Pierce and Benacerraf, 1969).
Analogous observations have been made by Oppenheim
et
al. (1968),
who found that adherent cells were required for antigen-induced lympho-
cyte transformation
in vitro. A
similar role for glass-adherent cells has
been claimed in lymphocyte transformation induced by PHA, or the
mixed lymphocyte reaction (Gordon, 1968; Levis and Robbins, 1969;
Levis
et
al., 1970). It should be stressed, nevertheless, that certain types
of immune responses, notably homograft reactions in
vivo
and
in oitro,
may not always depend on the presence of macrophages during the
inductive stage (Gowans, 1965; Strober and Gowans, 1965; Wilson,
1967) . Adherent cells, moreover, may well comprise cell populations
other than macrophages (Bianco
et al.,
1970).
The immunogenicity
of
several protein antigens is directly related
to their state of aggregation and, therefore, to their relative palatability
to macrophages. Rapidly sedimentable bovine y-globulin was immuno-
genic in mice, whereas supernatant fractions induced tolerance ( Dresser,
1962; Claman, 1963). Similarly, biologically “filtered,” isotopically labeled
BSA, which had been passaged through rabbits, was recovered from
their serum and found to be tolerogenic when injected into further re-
cipients, Equivalent amounts of unfiltered BSA were immunogenic ( Frei
et
al., 1965). Again, in contrast to the highly immunogenic native anti-
2.
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
287
gen, cyanogen bromide-digested fragments of Salmonella adelaide flag-
ellin were sho wn to
be
tolerogenic in a du lt rats ( C. R. Parish e t al., 1967) .
Such observations, together with
the
finding that some macrophage-
associated antigens may possess increased immunogenicity (see below),
have led to the formulation of
a
macrophage bypass theory of immuno-
logical tolerance, which has been amply reviewed by Leskowitz (1967)
an d by Dresser an d Mitchison (19 68). On th e other han d, efficient phago-
cytosis cannot be the sole prerequisite for immunogenicity. Undigestible
or slowly metabolized antigens, such as pneumococcal polysaccharides
(Fel ton
et
al., 1955; Coons, 19f33) or copolymers of D-amino acids (C ar -
penter et
al.,
1967; Janeway an d Hu mp hrey , 1968, 1969 ), are very effici-
ently taken up by macrophages but tend to induce paralysis rather than
immunity (Fel ton e t al., 1955; Janeway an d Sela, 1967; Janeway an d
Hu mp hrey, 1969; How ard a nd Siskind, 196 9).
Several proteins have been found to be more potent immunogens
when administered to recipients within macrophages than when given
in free form (Mitchison, 1967, 1968, 1969a; Un anu e an d Askonas, 19 6t h) .
This difference, however, seems to hold only for antigens which are
rather slowly taken up by phagocytic cells
in vivo.
In contrast, the im-
munogenicity of aggregated materials was not further increased after
ingestion by macrophages
in
vitro
( Mitchison, 1969a).
Finally, one of the more challenging claims for
a
macrophage
step in the induction of immunity stemmed from the observation that
filtrates and HNA-containing fractions of macrophages incubated with
T, bacteriophage or heinocyanin may have elicited specific antibody
formation both in v i v o and in vitro (Fish ma n, 1959, 1961; Fishman an d
Adler, 1963, 19 64 ). Th e presence of specific antigen in such m acrophag e
RNA preparations was later demonstrated and confirmed (Askonas and
Rhodes, 1965; Fried m an
e t
al., 1965; Adler et al . , 1966). The significance
of antigen-RNA complexes is now un de r intensive investigation
(
Gottlieb
et al., 1967; Gottlieb, 1968, 1969a,b; Gottlieb and Straus, 1969).
In summary, although there is a considerable body of evidence im-
plicating macrophages in th e afferent limb of th e imm une response, their
exact role is by
no
means clear. Macrophages may simply prevent excess
antigen from paralyzing lymphocytcs, act as convenient carriers of
antigen throughout the body, concentrate and retain antigen over longer
periods of time, present antigen to responsive cells, degrade antigen into
active subunits, convert crude antigen into a better immunogen, trans-
late antigen into specific information, or, finally, exert some nonspecific
effect on antigen-reactive cells which allows them to perform properly.
Moreover, these mechanisms are by no means mutually exclusive.
3.
4.
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288 GERALD WEISSMANN AND
PETER
DUKOR
B. THE LOCALIZATIONF ANTIGEN
N LYMPHOIDISSUES
1.
hlacrophages
and
Dendritic Reticular Cells
Depending on the route of injection, antigen is cleared from the
bloodstream, serous cavities, or lymphatic channels, and the bulk of the
material is taken up by free or fixed macrophages throughout the body
(Sabin, 1939; Kruse and McMaster, 1949; Coons
et
al., 1951). Antigen
concentration in phagocytic cells of liver, lung, and bone marrow may
far exceed the uptake by macrophages in lymphoid tissues (Campbell
and Garvey, 1961, 1963; Ada
et al.,
1964b), but it is obvious that the
functional importance of the different subpopulations may vary tre-
mendously. This view
is
supported by the studies of Franzl
(1962)
and
Cohn (1964), who demonstrated considerable differences in the reten-
tion of immunogenic material between liver and spleen and between
alveolar and peritoneal macrophages. It was calculated, moreover, that
only a minute proportion (less than 0.05% in the case of keyhole limpet
hemocyanin) of the originally administered antigen played a significant
role in the actual process of immunization (McConahey
e t
al.,
1968). It
is crucial for the understanding of the mechanism of antibody forma-
tion to define the anatomical pathways
of this fraction
through lymphoid
tissues.
When apparently nonantigenic materials, such as titanium dioxide, iso-
topically labeled chromium phosphate, Thorotrast, saccharated iron
oxide, or colloidal carbon, were injected intravenously into rats, mice,
or rabbits, they quickly became localized in macrophages of the red pulp
of the spleen-particularly in the marginal zone surrounding the follicles
of the white pulp (Goulian, 1953; Baillif, 1953; Odeblad
et al . ,
1955;
Nossal
et al.,
1966; Pinniger and Hutt, 1956; Hunter and Wissler,
1965; Hunter
e t al.,
1969). In Nossal’s study, some of the carbon was also
found in “tingible body macrophages”
of
the secondary foIIicIes. Simi-
larly, injection of substances with little or no antigenicity (gelatin, rat
hemoglobin, or rat red blood cells) into the footpads of rats was associ-
ated with uptake of the material in the corresponding locations of the
draining lymph nodes, i.e., lining macrophages of the medullary and
marginal sinuses (Ada
et al.,
1964a). Also, if antigens, such as horse
ferritin, BSA, diphtheria toxoid,
Salmonella adelaide
flagella, (T,G ) -A-L,
hemocyanin, or HSA, were administered to nonimmune recipients, they
were always found to be concentrated first in the sinus lining macro-
phages of lymph nodes or in the marginal-zone macrophages of the spleen
(Ada
et al.,
1964a; Nossal
e t
al . , 1964, 1966; McDevitt
e t
al., 1966;
Humphrey
et
al . , 1967; Humphrey and Frank, 1967). Later, antigenic
material appeared to associate with surfaces of nonphagocytic, dendritic
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
289
reticular cells in follicles
of
lyniph nodes and spleen (see also White,
1963
) . These elements could be distinguished clearly by morphological
and functional criteria from macrophages
(
Mitchell and Abbot, 1965;
Nossal et
al.,
19 68 b) which are also prcsent in th e cortex of lym ph
nodes and in the follicular areas of the spleen,
Follicular antigen localization was the subject of a recent review
artice by McD evitt (19 68 ). It seemed clear tha t specific antibody
was required fo r efficient trap pin g of antigen on t he surface of dendritic
reticular cells. In most cases, follicular localization coincided with the
appearance of circulating antibody ( Hum phrey an d Frank, 1967; W hite
e t al., 1967; French et
al.,
19 69), was more pronounced in actively or
passively immunized animals (Nossal
et
al.,
1965b; M cDevitt
e t
al.,
1966;
Mitchell and Abbot,
1965) , and could be reduced or abolished by
irradiation (Jaroslow and Nossal, 1966; Williams, 1966b), chronic
thoracic duct drainage (Williams, 1966a),
or
induction of specific im-
munological tolerance (H um ph rey an d Frank, 19 67) . Not surprisingly,
germfree rats had a diminished capacity for localization of antigen
on dendritic cells (J. J. Miller
et
ul., 1968). Claims that nonantigenic
material can also be localized on dendritic reticular elements of lymph
node follicles (Cohen et
al.,
1966; Hu nter e t al., 1969) have been open
to criticism (Nelson, 1969) and await further confirmation.
Tr ap pin g of antigens b y reticular cells may not be the only mechanism
responsible for their localization in follicles. Recently, there has been
characterized a new subpopulation of lymphocytes which binds antigen-
antibo dy complexes in the presence of modified complemen t Bianco
et
al.,
19 70 ). Since th e bulk of this subpo pulation, terme d “comple-
ment receptor lymphocytes,” resides in the follicular areas of peripheral
lymphoid tissue (Dukor et al., 19 70), i t would seem likely th at the y
contribute to antibody-mediated retention of antigen by lymphoid
follicles. Follicular antigen localization is thought to provide the sub-
strate for the firing of secondary immune responses (Ada et al., 1968) ,
long-term antibody production, and the proliferative expansion of im-
munologically committed cells (Hanna
et
al., 19 69 ). I t is not, however,
a prerequisite for th e induction of a primary response.
2 .
Antibody-Producing Cells
The presence of antigen in antibody-producing elements has been
the subject of controversy
(
McD evitt, 19 68 ). Em ploying highly sensi-
tive electron-microscopic and radioautographic techniques, several au-
thors have followed th e fate of ferritin an d isotopically labeled Salmonella
flagellin, (T,G )-A -L, hemocyanin, an d HSA in dig eren t species. Th ey
have failed to demonstrate any detectable antigen outside macrophages
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290
GERALD WEISSMANN A N D PETER DUKOR
or dendritic reticular cells (de Petris and Karlsbad, 1965; Buyukozer
e t al.,
1965; Nossal
e t
al., 19665a; McDevitt
et
al., 1966; Humphrey and
Frank,
1967).
Other workers have reported considerable amounts of
antigen in plasma cells, their precursors, and even in small lymph node
lymphocytes, both of unprimed animals (Roberts,
1966;
H a n a nd
Johnson, 1966; Han et al., 1967) and to a greater degree, of specifically
immunized animals ( Wellensiek an d C oons, 1964; Roberts, 1 964). In a
reevaluation of this problem, Nossal
et
al. (1967) were able to t race
labeled flagellin in a minority of isolated single antibody-forming cells
obtained from lymph nodes very early during primary and secondary
immun e responses, bu t never in thoracic d uc t lymphocytes. Th e authors
concluded that antigen
could
enter primitive plasmoblasts, but that
it
was impossible to decide whether antigen entry was necessary for cell
proliferation and antibody production.
Recent findings challenge the belief that phagocytic cells are in-
capable of antibody synthesis. Hannoun and Bussard (1966) and Bussard
and Lurie (1967) identified large cells, apparently histiocytes with
vesicular cytoplasm, as antibody producers in u i t ro . Pernis e t al. (1966)
described polarized epitheloid cells, obtained from granulomatous lesions,
which shared the ultrastructural characteristics both of macrophages and
of plasma cells. Holub
e t al.
(1966) and Holub and Hauser (1969)
demonstrated specific, puromycin-sensitive, hemolytic-plaque formation
by aveolar histiocytes containing abundant vesicles, lysosomes, and in-
clusion bodies. Unlike lymphoid cells which produced antibody, many
of these cells were destroyed by the uptake of silica. Moreover, plaque-
forming spleen cells were recently shown to incorporate carbon particles
(No ltenius an d Chahin, 19 69). It is not clear whether these findings
indicate that primitive macrophages possess the capacity to synthesize
specific antibody. Indeed, the data can also be interpreted to demon-
strate that activation of the vacuolar apparatus follows exposure of
lymphocytes to antigen or nonspecific mitogens (R . Hirschhorn e t
al.,
1967; Brittinger
e t
nl., 1968) (see be low).
C.
THE
FATE
F
ANTIGEN
N
MACROPHAGES
1 .
Endocytosis
Cellular ingestion of particulate and soluble foreign material occurs
by phagocytosis and pinocytosis. The histochemical, ultrastructural, and
metabolic features of this process have been reviewed by Cohn (1968),
Daems
et al.
(1 96 9), an d Jacques (19 69) . Cell eating and cell drinking
are basically similar events involving the invagination of plasma mem-
brane which fuses with itself and thus interiorizes extracellular com-
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THE ROLE OF LYSOSOMES IN IMMUNE
RESPONSES
291
ponents within a phagosome or pinosome. However, the metabolic re-
quirements of phagocytosis and pinocytosis, or rather, of the uptake of
extracellular material by large and by small vesicles into mononuclear
phagocytes, may differ considerably ( Cohn, 1968; Casley-Smith, 1969).
The rate of endocytosis can
be
enhanced by a variety of stimulants.
Increased phagocytic capacity of macrophages in vitro was induced by
maintenance of the cells over prolonged periods in culture (W.
E.
Bennet an d C ohn, 1966; Perkins e t a t , 1967) or through nonspecific acti-
vation by BCG (Ev ans and Myrvik, 19 67 ). Pinocytosis w as stimu lated by
anionic molecules ( Cohn and Parks, 1967a )
,
by nucleosides and nucleo-
tides (C oh n and Parks, 1967 b), an d by antibody directed against
a
macrophage membrane ant igen (Cohn and Parks , 1967~) .
Although specific antibody is not an absolute prerequisite for the in-
gestion of all types of foreign material by macrophages, recognition
phenomena are involved in the initial attachment phase preceding en-
gulfment ( Rab inovitch, 1967a,b, 1968, 19 69 ). Special significance may
be at tr ibuted to a macrophage membrane coat that is sensitive to trypsin
(Vaughn, 196 5), chyniotrypsin, an d papain (Lagunoff, 196 9). This
coat has, ind eed , been shown to determine the attachment of d am ag ed
erythrocytes and the rate of pinocytosis of foreign protein. The surface
properties
(
Rabinovitch, 19 69 ), state of aggregation, a nd relative
foreigness (Perkins and Leonard, 1963) of the offered material are
also decisive for its palatability to macrophages. The latter phenomenon,
however, may reflect an involvement of “natural antibodies.” The im-
portance of cytophilic antibodies, complement factors, and opsonins for
uptake of foreign matter by phagocytic cells was amply dealt with in
a
recent review (N elso n, 1969) an d is beyon d t h e scope of this article.
2.
Segregation
of
Foreign
Material within
Lysosomes
The current concepts of granule 00w and merger during endocytosis
ar e based on light microscopic an d ultrastructural observations on macro-
phages from different sources (Essn er, 1960; Novikoff a nd Essner,
1960; Cohn and Wiencr, 1963a,b; Cohn
et
nl., 1966; Lea ke a nd Myrvik,
1966; North, 1 96 6). Newly form ed cnd ocytic vacuoles, which ar e prob-
ably devoid of acid hydrolases, flow from th e plasma m em bra ne to-
ward the Golgi region where they fuse with primary lysosomes. The
extrusion
of
granule contcnts into the phagosome is evidenced by the
loss of primary lysosomes from the cytoplasmic matrix and the appear-
ance of hydrolases in the phagolysosomcs
(
Straus, 1 96 4) . These digestive
bodies ( also called “heterophagic vacuoles” or “sccondary lysosomes” )
probably display decreased resistance to mechanical stress, which may
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TABLE I
EXAMPLESF SEGREGATIONF FOREIGN
ATERIAL
N MACROPHAG
Foreign
material
Source
of
Techniques
macrophage usedo
Thorium dioxide
Plutonium dioxide
Gold
Triton
WR-1339
Various oligo- and polysaccharides
Dextran
Various synthetic oligopeptides
Poly-(D-tyrosine, D-glutamic acid,
Poly- (btyrosine, Irglutamic acid)
Horseradish peroxidase
Bovine serum albumin
D-alanine)
Human serum albumin
Rat liver
Mouse spleen
Rabbit liver
Rat peritoneum
Mouse peritoneum
Rat liver
Rat spleen
Mouse peritoneum
Mouse
spleen
Rat spleen
Mouse peritoneum
Mouse peritoneum
Mouse peritoneum
Ra t liver
Guinea pig peritoneum
Mouse liver
Mouse peritoneum
Rat lymph node
Rat lymph node
Mouse peritoneum
Mouse peritoneum
EM
EM, HC
DC,
DG
EM
LM
DC, DG, EM
DC, DG
LM, EM
EM, HC
DC, DG
LM, RA
DG
DG
LM, HC
EM, HC
DC
DG
DC
DC, DG
LM, RA
LM, EM, RA
Wiener et a
Daems and
Weissman
Sanders an
Cohn and
Wattiaux e
Bowers an
Cohn
and
Daems and
Bowers an
Ehrenreich
Kolsch and
Kolsch and
Straw,
196
Catanzaro
Mego and
Kolsch and
Ada and L
Williams a
Ehrenreich
Rhodes
et
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Ferri t
i i
R a t lymph node EM Buyukozer
Mouse peritoneum LM, EhI Rhodes
et
a
M a i a syuinado
hemocyairin Ra t lymph node DC Ada and L
hiouse lymph node DC, DG Askonas et
Rabbit spleen DC Uhr and W
Rabbit liver DC, DG Weissmann
Bacteriophage T2 Rabbit peritoneum
EM
Aronow el
Ra t peritoneum
EM
Friend
et a
Bacteriophage
OX 174
Guinea pig liver DC Vhr and W
Salmonclla
arlelaidr flagella Rat lymph node DC, DG Ada and W
Salmonella
adelaide
flagellin Ra t lymph node DC Ada and L
Williams a
Esehcrzchia
coli
Rabbi t lung and DC Cohn,
1964
peritoneum
DC Franzl, 196
Mouse a i d ra t red blood cells Ra t spleen DC, DG Bowers an
DC = differential centrifugation HC
=
histochemistry
Rabbit liver DC, DG Weissmann
Ra t lymph node EhI, R A Nossal
et a
Ra t lymph node and DC, DG
spleen
Listeria
monocytogenes hlouse spleen Ehf Armstrong
Sheep red blood cells
Mouse spleen
a Key to abbreviations:
DG
=
density gradient analysis
EM =
electron microscopy
L N
=
light microscopy
R S
=
radioautography
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294 GERALD
WEISSMANN AND PETER DUKOR
account for the intracellular redistribution of lysosomal enzymes from
sedimentable to nonsedimentable forms during active endocytosis (Cohn
an d W iener, 19 63 b) . Uptake an d segregation of antigens within lyso-
somes have now been firmly established (Table I ) .
a . Nonantigenic Particulates. In ultrastructural studies, indigestible
inorganic particulates, such as thorium dioxide, plutonium dioxide, or
gold, were sequestered into phagolysosomes of Kupffer cells (Wiener
et aZ., 1964 ), spleen red pulp m acrophages (D ae m s and Persijn, 1964 ),
or mononuclear phagocytes of th e peritoneal cavity (C o h n an d Benson,
1965; Sanders an d Adee, 19 69 ). For weeks they rem ained associated
with increasingly smaller an d den ser lysosomes which assumed th e ultra-
structural characteristics of residual bodies. Intravenous injection of
thorotrast also resulted in an augmented density of hydrolase-rich
granules from rabbit liver homogenates (Weissmann and Uhr, 1968).
This new class of thorotrast-containing lysosomes could be separated
from other large granules and appeared to possess the characteristic
fragility of secondary lysosomes.
Triton WR-1339, a detergent, was also found to
be
sequestered by
lysosomal fractions obtained from liver (W attia ux et al. , 1963) an d spleen
(Bowers an d d e D uv e, 19 67 ). I n this case, th e secondary lysosomes be-
came less dense and, again, were more fragile to mechanical trauma than
acid hydrolase-rich granules from control animals.
Oligo-
and Polysaccharides. A number of indigestible di-, tri-,
and tetrasaccharides were readily taken up by macrophages in vitro and
became associated with swollen, phase-lucent, lysosomal storage gran-
ules ( Cohn an d E hrenreich, 19 69). In the electron-microscopic studies
of Daems and Persijn (1965) and Daems et al. (1969) , uptake of
dextran particles by phagosomes and their subsequent merger with
acid phosphatase-containing lysosomes could also
be
visualized. In a den-
sity gradient analysis of subcellular fractions from spleen homogenates
of 14C-dextran-injected rats, Bowers an d d e D uv e (19 67) demon-
strated localization of the label in the dense macrophage lysosome frac-
tion. Subsequent electron-microscopic examination of red pulp macro-
pha ges revealed selective association of dex tran with e lectron-dense
lysosomes containing abundant iron-rich breakdown products
(
Bowers,
It wou ld seem, therefore, tha t administration of a pp ro pr iat e materials
may change both morphological and sedimentation characteristics of
secondary lysosomes and thus provide a convenient label for lysosomal
subpopulations.
c .
Peptides and Proteins. Synthetic oligopeptides
(
Ehrenreich and
Cohn, 1969) a nd polypeptides (Kolsch a nd Mitchison, 196 8), serum
7,.
1969).
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
295
albumin (M eg o an d M cQueen, 1965; Ada and Lang, 1966; Mego
e t
al.,
1967; Ada, 1967; Williams and Ada, 1967; Ehrenreich and Cohn, 1967;
Kolsch and Mitchison, 1968; Rhodes
et
al.,
19 69 ), horseradish peroxidase
(S traw , 1964; Catanzaro
et
al., 1969), ferritin (Buyukozer e t al., 1965;
Rhodes et al., 196 9), and hem ocyanin (A da and Lang, 1966; Askonas
et al., 1968)
were all locali7ed within phagolysosomes.
When mouse macrophages were exposed to two antigens : isotopically
labeled HSA an d unlabeled ferritin at th e same time, both b ecame seques-
tered within th e same phagolysosomes (Rh ode s e t al., 1969). Similar
findings were obtained by Casley-Smith
(
196 9). This corroborates th e
results of an earlier study which demonstrated uptake of two separate
bacteriophages by a single, thorotrast-laden population of secondary
lysosomcs (Weissmann an d Uhr, 196 8). Ada and Lan g 1966) investi-
gated the subcellular distribution of HSA and hemocyanin in rat lymph
node ho mogenates af ter local administration of th e antigen. Both proteins
became partly associated with the large granule fraction where they were
degraded to low molecular weight products. Localization in this fraction
was considerably increased when antibody-complexed albumin was
injected. In subseque nt studies (W illiams an d Ada, 1967) such complexes
were shown to localize on dendritic reticular cells of lymphoid follicles,
whereas heat-denatured HSA became exclusively sequestered into vac-
uoles of medullary macrophages. Macrophagc-localized antigen banded
in urografin gradients together with latent lysosomal enzymes, whereas
antigen from lymphoid follicles was found to sediment at high density
values. This result suggests very strongly tha t a t least pa rt of the antigen
localizing in the “large granule” fractions from draining lymph nodes
was associated with m emb ranes from de nd ritic reticular cells or “comple-
ment-receptor lymphocytes.”
Experiments by Kolsch and Mitchison (1968) indicate that proteins
which have been ingested by macrophages are rapidly segregated into
two subcellular compartments with greatly differing characteristics.
Using a pulse-and-chase technique, the authors investigated the sub-
cellular distribution of
I T
and 13’I-labeled, heat-denatured BSA ( and
other protein antigens ) in homog enates of mouse peritoneal macrophages.
It was found that, irrespective
of the antigen dose used, 90% of newly
phagocytized label localized initially in a “turnover” compartment with
a density of 1.19 gm ./cm.3 This an tigen was rapidly broken down within
the next few hours. About 10%of the antigen, however, was associated
with a “storage” com partm ent con taining particles of higher density
(1 .2 s g m . / ~ r n . ~ ) .abel in this second localization was retained for many
hours. Although ingestion of antigen was barely altered by previous
X-irradiation of m acropha ge donors w ith 900 r, transfer of antige n into
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296
GERALD WEISSMANN
AND PETER DUKOR
the “storage” compartment seemed to be impaired. Antigen in the light
particulate fraction was readily solubilized by detergent and could be
precipitated by specific antiserum. Label in the dense fraction, on the
other h an d, resisted solubilization by m ost detergents a nd was apparently
membrane-bound. Essentially similar features were established for the
other antigens used, except that the slowly metabolizable synthetic poly-
peptides D-TGA and L-TG were initially associated with a still lighter
granule fraction before moving to the 1.19 “turnover” com partment.
As
judged by the enzyme distribution pattern, the two light frac-
tions--containing specifically precip itated antigen-were rich in lyso-
soma1 hydrolases. T h e na tu re of th e “storage” comp artm ent is less clear.
Antigen may become associated with very dense secondary lysosomes or
residual bodies. Alternatively, membrane-associated antigens may sedi-
ment together with the heavier, nuclear-debris fractions. This possibility
will be discussed further in the context of findings by Unanue and
Cerottini
(
1969) (se e be low ). Unfortunately, th e imm unological relevance
of the two compartments described by Kolsch and Mitchison remains in
doubt, since all of the subcellular fractions described proved to be very
poorly immunogenic when tested in a highly sensitive
in
vivo system.
d. Viruses.
Interactions between viruses and lysosomes were the
subject of a review by Dales (19 69 ). Fe w studies on macrophages have
been conducted at the subcellular level, and the role of lysosomal uptake
for the uncoating of the viral genome has yet to be clarified. Neverthe-
less, the fate of bacteriophage inside the vacuolar system of mononuclear
phagocytic cells ha s been investigated by several workers, since antibody
formation against these viruses has served as a choice model in im-
munology.
Electron microscopy of in
vitro
endocytosis of T2 phage by rabbi t
peritoneal macrophages revealed that virus particles were first absorbed
to the cell mem brane, then surrounded by macrophage pseudopods, an d,
within minutes, incorporated into phagocytic vacuoles. The phagosomes
then moved centripetally towards the perinuclear region. Indirect evi-
de nc e for fusion of phagosom es a n d depletion of p rimary lysosomes was
also obtained (Aronow et al., 1964; Friend e t al., 1969 ). In contrast to
preliminary observations by Fishman et al. (1 96 5), phage particles were
never detected over the nucleus or outside vesicular, membrane-bounded
structures in these studies.
Sequestration of intravenously injected bacteriophages T2 and +X 174
into the large granule fractions
of
guinea pig liver and ra bbit spleen, but
not of rabb it kidney, was dem onstrated in a stud y by U hr an d W eissmann
( 19 65 ). They assumed th at phage-containing lysosome fractions we re
derived from reticuloendothelial cells in these organs. Indeed, plaque-
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THE
ROLE
OF LYSOSOMES IN IMMUNE RESPONSES
297
24
20
forming phage could be released from the hydrolase-rich fraction by
lysolecithin. In further experiments, lysosome-rich fractions from livers
of rabbits injected with bacteriophage
T2,
+X
174,
and
F,
were purified
on sucrose density gradients. The phage particles sediniented together
with a hydrolase-rich fraction following administration
in
uiuo, whereas
their sedimentation properties proved to
be
different when phages were
admixed to liver homogenates
in
vi tro . Moreover, when Thorotrast injec-
tion was followed by the administration of bacteriophages, plaque-form-
ing units were associated not only with the original lysosomal popula-
tion, but also with the Thorotrast-induced, dense particles (Fig. 1) . t
therefore appeared likely that a population of secondary lysosomes par-
. Contro l Thor ot ros t
p -
glucuronidose
--
Ary lsu l fo tose
;
I
ZI
c
E
-
E
ZI
c
-
c
I-
s
LL
a
-
c
t
s
hcter iophage F
Injected 24 hrs after
o
horotrast, distribution
18hrspmva
injection
Tub e number Tube number
FIG.
1.
Distribution of bacteriophages and lysosomal hydrolases in large-granule
fractions of rabbit liver. Discontinuous sncrose gradient: tubes 1-4 = 1.8
M ;
5-8 =
1.7
M ; tubes 9-12
=
1.6
M ;
tubes 13-16 =
1.5M ;
tubes 17-20
=
1.4
M
sucrose. Up-
per left-distribution of p-glucnronidase and arylsnlfatase activities of large-granule
fraction from control aniinal; upper right+listribution of p-glucuronidase and aryl-
snlfatase activities of large-granule fraction from aniiiial injected 24 honrs previously
with colloiclal thorium dioxide
(
Thorotrast
);
lowcr Icft-clistribution of bacteriophage
Fs
in large-granule fractions, after aclniixture in vitro
18
hours after injection; lower
right-tlistribiitioii of liacteriophage
F2
18 hours after injection into rabbit injected
24 honrs previously with Thorotrast ( Weissinanii and Uhr, 1968).
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298
GERALD
WEISSMANN AND PETER DUKOR
ticipated furth er in the up tak e of newly ingested m aterial (W eissmann
and Uhr, 1968).
e. Bacteria and Bacterial Components.
Incorporation of whole
bacteria and bacterial antigens into phagolysosomes of macrophages has
been firmly established by many investigators (for review, see Allen,
19 69 ). Locally injected
Salmonella adelaide
Aagella is found within
the phagolysosomes of medullary macrophages from draining lymph
nodes, as shown by subcellular fractionation and electron-microscopic
radioautography. Soon after administration of iodinated flagella, most of
the radioactivity was foun d in th e supe rna tan t fraction of lymph n od e
homog enates (A d a an d Williams, 1966; Ada, 1 96 7). After
1
to 2 days,
however, about half of the radioactivity was present in the large granule
fraction, and
15%
f the label became associated with the nuclear-debris
fraction. Within the following 2 months, the proportion of radioactivity
in this latter fraction rose steadily to a bo ut 25%, wh ereas most of th e
remaining label stayed with the large granules. The antigen (reactive
with antibody) in the large granule residue was shown to become more
tightly associated with membranes.
It
is possible that antigen in this
and in the nuclear-debris fractions is associated with membranous ma-
terial derived from dendritic reticular cells or “complement-receptor
lymphocytes” which
so
avidly attra ct antigen-antibod y complexes.
High-resolution radioautography
( Nossal
et
al., 1968a) revealed that
iodinated flagella entered into macrophages by two pathways: some of
the IabeIed antigen was taken up by pinocytosis, whereas another frac-
tion seemed to penetrate the plasma membrane directly. Single grains
found lying, apparently free, in the cytoplasm up to 30 minutes or
longer were then surrounded by small dense vesicles and, subsequently,
sequestered into membrane-bounded inclusions. Evidence for fusion of
antigen-laden vacuoles with electron opaque primary lysosomes was
also obtained. Some of these antigen-containing bodies were very dense
and closely resembled the telolysosomes described by Gordon e t aE.
(1965).
As time progressed, many of the labeled phagolysosomes became
larger and more complex. As in the fractionation studies by Ada, radio-
activity remained associated with such lysosomes for at least 6 weeks.
A t no
time could significant labeling be demonstrated in nuclei, nor was
evidence obtained for the exit
of
labeled antigen fragments froin second-
ary lysosomes. Finally, entry of whole bacteria, e g , Listeria monocyto-
genes
( Armstrong and Sword, 1966) or
Escherichia coli
( Casley-Smith,
1969) into phagosomes and phago1ysosome.s of macrophages has been
visualized by electron microscopy.
f . Metazoczl Cells. Phagocytized metazoal cells, such as erythro-
cytes, are also sequestered into lysosomes. Franzl (1962) and Franzl and
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THE ROLE
O F
LYSOSOMES IN IMMUNE RESPONSES
299
Morello ( 1965) demonstrated the specific immunogenicity of lysosomal
fractions, which had been isolated from splecn homogenatcs of mice
injected with sheep red blood cclls
in
wivo.
W lien 51Cr-labeled erythro-
cytes wcre administered to rats, radioactivity could be recovered in
spleen homogenates. The label scdimented with the light lysosome frac-
tion
of
lymphoid tissue macrophages (Bowers and de Duve, 1967).
The data reviewed in this section indicate clearly that a wide range
of materials differing in size, physicochemical properties, and anti-
genicity become segregated by the vacuolar system. Whether antigens
can totally resist intralysosomal degradation, whether
a
fraction of the
ingested antigen can escapc from the lysosomal compartment, or else,
whether some
of
the ingested antigen is channeled into alternative path-
ways without ever e nte ring the vacuolar system is less clear.
3. Intralysosomal Degrnclution
a.
Oligosacchurides and Oligopeptides.
Once inside the vacuolar
system, ingested matter is rapidly broken down by hydrolases. In a s tudy
of
the uptake and intracellular hydrolysis by mouse macrophages of
carbohydrates, Cohn and Ehrenreich ( 1969) demonstrated that sucrose
a nd
a
number of other di-, tri-, and tetrasaccharides could not be de-
graded by macrophage lysosomal enzymcs. Consequently, these were
retained inside swollen storage vacuoles over long periods of t ime. In
such vacuolated cells, exogenous invertase and suitable hexosidases were
promptly takcn up by pinocytosis and found specifically to reverse lyso-
soma1 swelling. Presumably beca use small molecules c an diffuse across
lysosomal membranes, vacuolization was not induced by indigestible
sugars of low molecular weight. Similar rcsults were obtained with
readily pinocytosed, indigestible D-oligopcptides ( Ehrenreich and Cohn,
1969) .
b. Proteins. Intralysosomal degradation
of
a variety of antigenic,
exogenous proteins has been followed by morphological and biochemical
methods. Within 2 to
3
days the histochemical reaction product of
systemically administered horseradish peroxidase gradually disappeared
from the phagolysosomes of Kupffer cells (Straus, 1 96 4). In analogous
experiments, Cohn and Berison (1965) demonstrated the loss of pino-
cytosed fluorescein-labeled proteins from cultured mouse macrophages.
Low molecular weight breakdown products of HSA a n d Muiu squi-
nado
hemocyanin were recovered in highest proportiolls from the large
granule fraction of draining rat lymph nodes within 2 to
3
days after local
injection of th e antigens (A da an d Lang, 19 66). Th e breakdown of such
proteins might be fairly complete. Isolated liver lysosomes containing
formaldehyde-treated
1311
HSA were found to release monoiodotyrosine
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300
GERALD W E X S S M A N N A N D
PETER
DUKOR
into t he surrounding medium ( Mego et al., 19 67 ). Both externally labeled
1251-HSA Ehrenreich and Cohn, 1967) and internally labeled "-rabbit
hemoglobin (Eh renre ich and Co hn, 1968) were lost rapidly from phago-
lysosomes of cultured peritoneal mouse macrophages. Label was quanti-
tatively recovered from the culture fluid and found to be associated with
single amino acids. Peptides of low specific activity or low concentration
would not have been detected in these experiments. Moreover, some 10%
of the radioactivity of BSA and about 40%of hemoglobin were still
present in the m acrophages 40 hours after the antigen pulse (Ehrenreich
and Cohn,
1968).
Similar observations were made with other protein
antigens: isotopically labeled Maia squinudo hemocyanin ( Unanue and
Askonas, 19 68 b), keyhole limpet hemocyanin
(
Unanue, 1969; Unane
an d Cerottini, 19 69 ), or BSA (Kolsch and Mitchison, 1 968) w ere all
rapidly lost from pulse-fed peritoneal macrophages, but in each case a
small proportion
(
3-10%) of the initial radioactivity remained associated
with the macrophages for a much longer time. From the experiments
of
Kolsch and Mitchison (1968), it would appear that such label was no
longer (if ever) localized in the bulk of macrophage lysosomes; findings
of Un anu e ( U na nu e and Askonas, 1968b; Unanue and Cerott ini , 1969)
would suggest, instead, that the retained antigen fraction was bound to
the plasma m embrane.
c. Viruses. Although the role of macrophages in virus infections
has been investigated frequently, (for a review, see Nelson, 1969), few
data are available which directly implicate the lysosomal system of these
cells. Uhr a nd Weissmann (1 96 5) reported a considerable decrease
in
the infectivity of large granule fractions from guinea pig liver homog-
enates within 48 hours after injection of bacteriophage +X 174. How-
ever, a similar
loss
of infectivity was observed in the postgranular
supernatant. In rat macrophages exposed to bacteriophage 7'2, most
ingested phage particles underwent intralysosomal degradation within
45 minutes after uptake (Friend et al., 1969) .
d .
Bacteria and Bacterial Components. The role of macrophage
lysosomes in the breakdown
of
bacteria and bacterial antigens is well
established (Allen, 1969). Degrad ation of G roup A streptococci in
phagolysosomes of mouse macrophages was followed morphologically
by Gill an d Cole (19 65 ), who demonstrated an early, partia l loss of a n
immunofluorescent complex from the bacterial surface and subsequent
segregation of fluorescent M-protein in macrophage vacuoles. LOW
molecular weight breakdown products of Salmonella adelaide flagella or
of soluble flagellin were localized in large granule fractions from lymph
node homogenates within
1
to 3 days following local injection of the
antigens (A da an d Williams, 1966; Ada and Lang, 1 96 6). As judged b y
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301
HE
ROLE
OF
LYSOSOMES
IN
IMMUNE RESPONSES
high-resolution radioautography, substantial amounts of labeled flagellar
material were detectable in phagolysoson~es or at least 6 weeks
(Nossal
et al . ,
1968a) .
The bactericidal effect of macrophagcbs could in some instances be
correlated w ith th e availability of lysosomal hydrolases. T he developm ent
of bactericidal activity against gram -negative bacteria in rat macrop hages
during fetal and early postnatal l ife (Karthigasu et al. , 1965) was paral-
leled by the development of acid phosphatase-positive granules in such
phagocytes, however, their endocytic activity remained unchanged during
the critical period of observation (R ead e, 19 68). In rabbits, B CG-induced
alveolar macrophages were found to contain much higher activities of
acid hydrolases than oil-induced mononuclear phagocytes from the peri-
toneal cavity ( Cohn an d Wiener, 1 963 a). Correspondingly, imm uno-
genicity of an Escherichia coli agglutinogen was almost completely
destroyed within 2 hours by alveolar cells,
but
not by the peritoneal
macrophage population
(
Cohn, 1964).
Ingested bacteria may themselves induce changes of the lysosomal
apparatus. Berk and Nelson
(
1962) observed lowered hydrolase activi-
ties in mouse macrophages which were infected with
Pseudomonus
aeruginosa. Chronic infection with relatively resistant strains of myco-
bacteria might finally exhaust the enzym e complement of macro phage
lysosornes (Allen et al., 1965; Merkal et al. , 1968). Conversely, certain
bacteria an d bacterial components-notably BCG an d endotoxins-were
shown to elicit in macrophages an increased content of lysosomal hy-
drolases (G ro gg an d Pearse, 1952; Suter an d H ulliger, 1960; Thorb ecke
et al. , 1961; Heise et
al.,
1965; Saito and Suter, 1965). The “immune
phagocyte” concept, which is in part based on such observations, has
been repeatedly th e subject of extensivc reviews (S ut er an d Raniseier,
1964; Rowley, 1966; Mackaness and Blanden,
1967;
Nelson, 1969).
This scanty evidence (Section II,C,3,a-d) does no t perm it definite
conclusions as to the ultimate fate of intralysosomal antigen. It is not
clear whether digestible foreign matter is uniformly degraded within
the lysosomal compartment or whether, upon entering a heterogeneous
granule population, fractions of antigen may be retrieved and spared
from u ltimate destruction. I t is clearer tha t certain niacrophage-associated
antigens can escape degradation for considerable lengths of time a nd th at
such antigen may remain within lysosomes.
4 .
Extralysosoinal Sites
of
Antigen Sequestration
Most investigators have failed unequivocally to demonstrate that
antigens can
be
sequestered outside the lysosomal system. Possibly the
lack of positive data may simply reflect the insufficient sensitivity of the
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302
GERALD WEISSMANN AND
PETER
DUKOR
systems employed for the detection of antigen. Nossal et al. (1968a)
calculated, for example, that when high-resolution radioautography was
employed for the detec tion of lZ5I-flage1la substitution r ate of one iodine
atom per m olecu le), th e appearance of one silver grain over a cell section
could have represented 4000 antigen molecules per macrophage.
Some significance should, therefore, be attached to recent investiga-
tions in which the intracellular fate of peroxidase was followed at the
ultrastruc tural level. T he system ma de use of the amplifying effect
of
an electron opaque, enzymatic, reaction product. Following administra-
tion of lactoperoxidase to mice, the tracer was detected not only in
lysosomes of Kupffer cells bu t also in the p erinuclear s pa ce and in th e
rough-surfaced endoplasmic reticulum ( Graham
et
aZ.,
1969). Similar
results were obtained by Catanzaro
e t
al. (1 96 9) , who exposed guinea
pig peritoneal macrophages to horseradish peroxidase
in
vitro. Within
15
minutes, tracer was detected in pinocytic vacuoles, but 2 5 3 0 % f the
cells also displayed marked enzymatic activity in the perinuclear space
and in the rough endoplasmic reticulum. There was little increase of
reaction product in this location and no change in the proportion of
cells containing extralysosomal peroxidase after prolonged periods of
incubation, although phagolysosomal sequestration of the antigen con-
tinued.
The possible spatial association of an exogenous, antigenic protein
with rough-surfaced endoplasmic reticulum is particularly interesting
in view of recent findings of Gottlieb (1969a) and Bishop and Gottlieb
(
196 9). These authors incu bate d peritoneal macrophages with an iso-
topically labeled, soluble, synthetic copolymer, L-GAT. Macrophage
extracts yielded RNA-antigen complexes,
a
part of which were bound
to monoribosomes. It is, therefore, possible that certain antigens may
bypass the lysosomal compartment and gain direct access to the ribo-
somes of the rough-surfaced endoplasmic reticulum, there
to
become
complexed to an immunogenic RNA. Such data require further analysis
and confirmation.
An altogether different lysosome bypass mechanism is suggested by
experiments of Unanue and Cerottini ( 1969). Peritoneal macrophages
which had been exposed to keyhole limpet hemocyanin retained a small
fraction
(3%)
f the antigen on the cell surface for a period of several
days. Plasma membrane-associated hemocyanin was neither ingested nor
catabolized, bu t could be rem oved b y trypsin. T he presence of surface-
associated antigen
was
demonstrated by specific binding of antibody to
the membrane and by high-resolution radioautography. Indeed, the im-
munogenicity of transferred live macrophages bearing hemocyanin on
their surface was abolished by passive administration of antibody.
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
303
It is not clear whether reactive antigen associated with the cell
surface can pass through the lysosomal compartment. Unloading of
secondary lysosomes is thought to occur mostly through membrane
transport or diffusion of small breakdown products. Although reverse
pinocytosis has been proposed to account for the release of hydrolases
from endocytic cells (see below), no convincing evidence for lysosomal
defecation by mononuclear phagocytes has yet been obtained (Cohn
and Fedorko, 1969).
5. Immunogenicity
of
Macrophage-Associated Antigen
T he possible immunological significance of small amo unts of a ntigen ,
persisting in lymphoid tissue for prolonged periods
of
time, has been
established by the early work of Haurowitz (1960) and of Campbell
an d Garvey (1961, 19 63 ). A fraction of ingested
Maia squinado
hemo-
cyanin may be retained by peritoneal macrophages for at least 72 hours
an d, upon transfer in s uita ble recipients, elicits
a
specific antibody
response ( Un anu e an d Askonas, 19 68 b). Protein antigens persisting in
irradia ted macrophages may retain their priming capacity for a period of
u p t o
3
weeks (Pribnow and Silverman, 1969). Although, as discussed
above, the immunogenic protein may be carried on the surface of the
macrophage (U na nu e an d Cerott ini , 1969 ), earl ier data suggested an
association of immunogenic antigen with lysosomal fractions. Large
granule preparations from spleen, but not from liver, of mice receiving
sheep red blood cells conferred specific antibody-forming capacity to
primed (Fra nzl , 1962) and unprimed (Fr an zl and Morello, 1965)
recipients. In either case, a critical time interval between injection of
the antigen and transfer of th e granule fractions was important. Analogous
findings were obtained by U hr an d Weissmann (19 65) , who found th at
large granule fractions from liver homogenates of guinea pigs were
specifically immunogenic. While the actual number
of
plaque-forming
units in the lysosomal fraction decreased, inimunogenicity increased.
In both series of experiments, plasma-membrane contaminants could
have sedimented together with the large granules.
Moreover, the pathways of antigen handling by macrophages may
depend on the type
of
antigen involved. The priming capacity of
Maia
squinado
hemocyanin (U na nu e and Askonas, 1968a) and of hum an a nd
bovine serum albumins
(
Mitchison, 1968, 1969a) when transferred in
live macrophages, was u p
to
a lo00 t imes greater than when the same
antigens were injected in their free form. No such enhancement was
found during the secondary response. Differences in priming ability of
macrophage-associated and free antigen were much smaller in the case
of lysozyme or ovalbumin ( Mitchison, 19 69 a). Highly immun ogenic
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304
GERALD
WEISSMANN AND PETER
DUKOR
antigens, such as heat-aggregated or guanidated BSA ( Mitchison, 1969a)
or keyhole limpet hemocyanin (Unanue, 1969) were all much less im-
munogenic when they were transferred inside of macrophages rather
than injected directly.
Finally, evidence is accu mulating tha t antigen-RNA complexes ob-
tained from macrophages incubated with T2 phages (Friedman
e t
al.,
1965; Adler et
al. ,
1966; Gottlieb
et
al., 1967; Gottlieb, 1968; Fishman an d
Adler, 1968; Fishman, 1969), Maia squinado hemocyanin (Askonas and
Rhodes, 1965 ), or synthetic polypeptides (Pin chu ck
et
al., 1968; Go ttlieb,
1969a ) may transfer immunological reactivity very effectively to both
in vivo a nd in vitro systems.
T he protein moiety of the imm unoge nic complex of T2-RNA di d not
contain m ore tha n thirty to thirty-five amino acids (G ottli eb an d Straus,
1969) but could effectively inhibit phage-neutralizing antibody induced
with the complete virus (Gottlieb, 19 69b ).
It
was thus demonstrated th at
preservation
of
the native tertiary structure of tail fiber antigen was not
necessary for the immunogenicity of bacteriophage T2. Nevertheless,
other antigens are known to elicit the formation of antibodies directed
against determinants associated with tertiary structure ( Kaminski, 1965).
These observations further suggest that antigen handling by macrophages
(a n d the vacuolar system) may differ considerably, depe nd ing upon
the antigen.
I l l
Mediators of Tissue In ju ry Found in Lysosomes
Mediation
of
immune injury
has
already been discussed extensively
for this series
by
Cochrane (1968). Since that survey, the functions of
leukocytes, and especially their lysosomes, have been investigated further.
Whatever the role of humoral m ediators in acu te inflammation, structura l
injury to tissues
(as
opposed to vasodilation, pain, and swelling) cannot
proceed without hydrolytic degradation of extracellular and intracellular
macromolecules. Lysosomes appear suitably equipped for this role.
Substances found in lysosomes of leukocytes or other cells have now
been shown capable of degrading the following materials relevant to
tissue injury: collagen, elastin, protein polysaccharides of cartilage, intact
cartilage, componen ts of th e complemen t system, hya luron ate, chondroi-
tin sulfates, endotoxin, histones, a nd nucleic acids. A sum ma ry of these
actions of lysosomal enzymes (w ith app ro pria te references) is found in
Table 11. One note of caution: it is still not entirely clear whether all, or
indeed most, lysosomal hydrolases can attack undenatured substrates at
hydrogen ion concentrations likely to obtain under physiologicil or even
pathological circumstances. The best example is the collagen molecule,
which in its native, und ena tured state is cleaved at neu tral p H by lyso-
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TABLE I1
HYDROLYSISF hfACROMOLECCLES RELEVANT
O TTSSUE
NJURY B Y LYSOS
Substrate Enzyme Source
Collagen
Protein polysaccharides
Protein polysaccharides
Protein polysaccharides
Hyalnronate, chondroitin
Cartilage matrix
Elastin
Arterial walls
Basement membranes
Erythrocyte membranes
sulfa1
Collagenase
Hyaluronidase
Neutral protease
Cathepsin D
Cathepsin
11
Elastase
Elastase
Cathepsins D, E
e
Hyaluronidase
>
Extracellular structures
Leukocytes
Liver
Leukocytes
Liver cartilage
Liver, bone
Cartilage
Leukocytes
Leukocytes
Lei ikocy
es
Liver
Intracellular structures
Mitochondria
?
Liver
11eoxyribonucleic acid Deoxyri honuclease
Liver, etc.
Ribonucleic acid
Itibonuclease Liver, etc.
Histones
Histonase
(pH
7.4) Leukocytes
Histones
Cathepsins D, E Leukocytes
C’ls,
C‘lS
C’3, C‘5
Kinins
Fibrin
Thyroglobuliii
?-Globulin
Endotoxin
“Cytotaxigens”
Plasminogen
Circulating materials
Neutral protease Leukocytes
Neutral protease Leukocytes
Acid peptidases Leukocytes
? Leukocytes
Cathepsins D, E Spleen
Neutral, acid proteases
? Neutral proteases Leukocytes, macrophages
Urokinase Kidney
Beef, human spleen
?
Liver
Weissm
Janoff
Lazaru
Weissm
Barret,
Aronso
Ali (19
Janoff
Cochra
Desai
Tappel
ernar
de Duv
P. Dav
P. Dav
Taubm
Taubm
Melmo
Riddle
Weigle
Fehr
et
Fdkins
Bore1 e
Ali and
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306 GERALD WEISSMANN AND
PETER
DUKOR
somal collagenase (Lazarus e t al., 1968) to yield two distinct subunits
corresponding to 25 a nd 75% of th e m olccule. Su bsequen tly, it m ay b e
attacked by less specific proteascs, such
as
those of macrophages
(Woessner, 1965 ). W he n native collagen is exposed to acid p H ,
it
becomes
partially denatured and susceptible to the latter group of enzymes. In
similar fashion, proteins such as PP-L
(
Weissmann and Spilberg, 1968),
thyroglobulin (Weigle et al., 1969), or histones may be denatured either
during isoIation or assay, so as to render them prey to the known lyso-
somal proteases, rather than to specific enzymes. Furthermore, a series
of nonenzymatic factors have been isolated from lysosomes. These ind uc e
capillary permeability (Burke e t al., 1964), release histamine from mast
cells (Janoff
e t
al.,
196 5), provoke fever (p yr og en ) (Herion
e t
al.,
1966) ,
an d kill bacteria (Zeya a nd S pitznagel, 19 66 ). I t is beyond th e scope of
this review to discuss these in detail; instead, we shall focus on the
mechanisms that govern their release in immune reactions.
IV.
Lysosomes i n Four Types
of
Im m u n e In j u r y
One convenient way of classifying immunologically induced tissue
injury is according to C oombs a nd Gel1 (1 96 3 ), who suggest four
categories into which such reactions can be placed. T y p e I reactions
are acute allergic reactions, mediated by
IgE
and the release of vaso-
active amines from basophiles and/or mast cells which have been pas-
sively coated by antibody. T y p e ZZ reactions are med iated by comp lement
after circulating antibodies have been attached to the surface antigens of
target cells. In T y p e
ZZI
reactions, circulating antigen-antibody complexes
activate complement in the fluid phase. T y p e ZV reactions ar e mediated
by lymphocytes that have become transformed and rendered capable of
tissue injury after
a n
encounter with antigen or foreign tissue. None of
these categories, of course, is either exclusive or comprehensive. Thus
anaphylaxis is mediated by both Type
I
and Type
I11
reactions, homo-
graft rejection can result from Type
I1
and Type
IV
reactions; etc.
Furthermore, autoimmunity is such a complex phenomenon that it may
well fall into all 4 categories. T he role of lysosomes in auto imm unity is dis-
cussed elsewhere ( W eissmann, 1964, 1965,1966) an d is no t detailed below.
A. TYPE REACTIONS
The cell types which are prominent in these reactions are the blood
basophile, the tissue mast cell, a nd t he eosinophile. From the work of
Terry et al. (1 96 9) , of Fedorko a nd Hirsch ( 1965) , and of Sampson and
Archer ( 19 67 ), clearly th e blood basophiles contain a uniform popula-
tion of granules, which resemble those of other polymorphonuclear
leukocytes only in their biogenesis from cisternae of the Golgi apparatus
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THE
ROLE OF LYSOSOMES IN IMMUNE RESPONSES
307
( Te r r y et al., 19 69 ). T he granules can b e shown to possess a crystalline
material disposed
as a
hexagonal latt ice, although
a
hexagonal array of
microfilaments is not excluded. The basophiles are poorly phagocytic,
and t heir granules contain few acid hydrolases, bu t a re rich in histamine.
There are, as yet,
no
compelling reasons to classify thc granules
as lyso-
somes. Mast cells, which bear some relationship to basophiles have been
more extensively studied. Their specific granules contain histamine,
serotonin, heparin, and a chyniotrypsin-like enzyme
(
Selye, 1965). T he
granules can be isolated in intact form without an adherent membrane
(Lagunof f
et al.,
1964); indeed, their membranes may form
a
sort of
cytoplasmic syncytium (Pa da w er, 196 9). Since
it
is presumed that the
overall integrity of mast cell granulcs is governed by electrostatic inter-
action, there is little reason to regard them as true lysosomes. The cells
ar e clearly phagocytic-they ta ke
up
colloidal thorium dioxide, zymosan,
etc.-but the merger of endocytic vacuoles with mast cell granules is
somewhat diffe rent from that described with tru e lysosomes (neu troph ile,
macrophages
) (
Padaw er, 1 96 9) . Eosinophiles con tain granules th at differ
only in enzym atic content (la ck of lysozyme, high peroxidase ac tivi ty)
from neutrophiles. Their granules are true lysosomes, and they merge
with endocytic vacuoles in a manner strictly comparable to that of the
neutrophile, after uptake of particles, cells, or immune complexes (Archer
and H irsch, 19 63a ,b). Th ey are attrac ted chemotactically to antigen-
antibody complexes
(
Lit t , 1964).
Indeed, it has been clearly shown that Type I reactions in vitro do
not involve lysosomes. Thus, Pruzanski a nd Patterson (196 7) stu died th e
subcellular distribution a nd release of histamine a nd lysosomal hydrolases
in hum an leukocytes. Both the amine a nd lysosomal 8-glucu ronid ase we re
found in large granule fractions. When leukocytes were obtained from
sensitized individuals and exposed to antigen
in vitro
histamine was
released into cell supernatants and surrounding media roithout con-
com itant release of @-gluc uronidase. In contrast, upta ke of sta rch pa r-
ticles was associated with release of p-glucuronidase, but not of histamine.
Crowdcr e t al. (1969) obtained similar dissociation between release of
histamine and two IysosomaI enzymes after challenge by opsonized
staphylococci or a protein antigen. W e have not only confirmed these
da ta bu t also demonstrated tha t procedures t ha t can raise the intracellular
level of cyclic 3',5'-adenosine mo nophosph ate c an inhibit release both
of histamine and of p-glucuronidase (May
e t al.,
1970) (Tables
I11
and
IV )
.
Th ese experiments indicate tha t the mechanisms th at govern release
of histam ine (fr om specific granules of blood b aso ph iles ) an d of p-glu-
curonidase
(
from azurophile granules of neutrophiles
) (
Baggiolini
et al.,
1969) can be affected by similar pharmacological means
(see
b e l o w ) .
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308
GERALD WISSMANN AND
PETER
DUKOR
TABLE 111
O N
RELEASE
F
HISTAMINE
ROM
HUMAN
OLYMORPHS
EFFECT OF THEOPHYLLINEN D / O R CYCLIC ADENOSINE3’$’-MONOPHOSPHATE
Agent added
_ _ _ ~ _____
No. of Total histamine
Experiments released (yo)
None 9
Zymosan
9
Ragweed
6
pg./ml.
6
Ragweed
+
theophylline
10-3 M
2
Ragweed + cyclic adenosine 2
Ragweed
+
cyclic adenosine
4
3’,5’-monophosphate M
3’,5’-monophosphate
M
+ theophylline
10-3 M
0-3
3 . 6
74.0”
14.G
60.5
O. Ob
0
p
(vs.
control)
< 0.01.
p
(vs. ragweed) <
0.01.
Mann-Whitney “U” test (modified
from
May et al.,
1970).
Moreover, they also clearly dem onstrate t ha t reactions m ediate d by Ig E
result i n histamine release w ithout an eff ect upon extrusion of enzyme
from lysosomes, whereas, phagocytosis has the opposite action.
These
considerations further remove the basophile granule from the general
category of lysosomes.
Oth er mediators
of
immediate reactions are the kinins (reviewed by
Kellermeyer and Graham, 1968) and SRS-A (Orange et al., 1969).
W hereas kinins are generated from plasma by neutral a nd acid
proteases, acting on circulating kininogen, the y can be degrade d by acid
TABLE IV
O N
RELEASE F ~-GLUCURONIDASEROM HUMAN
OLYMORPHS
I~FFECTF THEOPHYLLINE A N D / O R CYCLIC ADENOSINE‘,5‘-MONOPHOSPHATE
~
Agent, added
No.
of
Enzyme release
Experimen t,s (pg.*/ml./hr.)
None 9
Triton
X-100
(0.2%)
9
Ragweed 6 pg./ml. 6
Zymosan 9
Zymosan + t,heophylline l O P
M
5
Zymosan
+
cyclic adenosine
3
Zymosan
+
cyclic adenosine 4
3’,5’-monophosphate lo-* M
3’,5‘-monophosphate M
+ theophylline
M
4 . 7
47.
Oa
5 . 0
1 3 . e
8.3b
12.2
5.2*
* p (vs. eont.ro1) < 0.01.
* p
(vs.
zymosan)
<
0.01. Manri-Whitney “U” test (modified
from
May et ul.,
1970).
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THE
ROLE OF LYSOSOMES
IN IMMUNE
RESPONSES
309
proteases (kininases ) of leukocyte lysosomes ( Melmon and Cline, 1968).
Hegner
(
1968) has found both kinin-forming and kinin-destroying
enzymes localized in lysosomal fractions
of
horse leukocytes. The SRS-A
appears to be formed by polymorphonuclear leukocytes ( Macmorine
et al., 1968;
Orange et al., 19 69 ), but i ts subcellular distribution has not
been studied extensively. Release of SRS-A from leukocytes can also be
influenced by agents which modify intracellular levels of cyclic AMP
(Ishizaka et al., 1970) .
Since the description by Janoff and Zweifach (1964) of a cationic
protein component of neutrophile lysosomes, capable of provoking capil-
lary permeability, several studies have implicated this and related pro-
teins in acute inflammation. This subject was reviewed by Cochrane
( 19 68 ). Briefly, the cationic proteins of neutrophiles ar e qu ite hetero-
geneous. One well-defined protcin fraction
(
Seegers and Janoff, 1966)
induces inflammation by virtue of the disruption of mast cells; other
fractions induce inflammatory responses by less well-defined means
(
Cochrane, 1968
)
.
B. TYPE 1 REACTIONS
When heterologous antibodies are added to living cells, the conse-
quences to lysosomes depend upon the presence of complement.
Thus,
Dumonde et al. (1965) added hetcrologous antibody to ascites tumor
cells in the absence of compleineiit an d fou nd ( b y histochemical m ean s)
alterations in the access of lysosomal acid phosphatase to substrate at
discrete granular sites in the cytoplasm. In t he presence of com pleme nt,
however, the granules were no longer observed. Cell death had been
accompanied by disruption
of
lysosomes, the enzymes of which became
diffusely distributed throughout the cytoplasm. Weiss and Dingle ( 1964)
raised antiserum in rabbits to lysosome-rich fractions
of
rat l iver. Heated
antiserum had no effect upon their test systems, i.e. lysosome-rich sub-
cellular fractions of rat liver, slices
of
rat liver, cultured fibroblasts. In
contrast, unheated serum induced release of lysosomal hydrolases from,
and altered the staining
of,
lysosomes, and released enzymes from intact
liver slices. Ho wev er, no effect
of
the serum upon isolated organelles was
de tec ted . Dorling an d Loewi (19 65 ) observed altered staining of lyso-
somes in rat kidney cells exposed to rabbit antisera. Fell and Weiss
(19 65) p repa red ra bbit antiserum against fetal mouse cells. Th e changes
induced by unheated sera in cultured mouse limb bones near term
resembled closely those provoked by other agents which caused release
of lysosomal enzymes from cells, i.c., rctinol, sucrose. Each of these ex-
periments suggested that complement-sufficient antisera directed against
components of cell membranes acted to “labilize” lysosomes by an
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310
GERALD WEISSMANN
AND PETER
DUKOR
indirect action me dia ted by primary events at th e cell surface. In contrast,
Quie and Hirsch ( 19 64 ), using purer, absorbed, heterologous antisera
directed against leukocyte lysosomes, were able to induce intact leuko-
cytes to degranulate-with ap pa re nt discharge of acid hydrolases into
cytoplasm. Such antisera provoked release of thre e lysosomal hydrolases
from isolated leukocyte lysosomes in
v i t r o .
This apparent discrepancy
awaits resolution. The group in Cambridge (Fell
et
al., 1966, 1969;
Dingle et at., 1967; Coombs and Fell, 1969) investigated in detail the
action of complenient-sufficient antisera upon cartilage and bone in
culture. Their results may be summarized as follows:
1. Both antifowl erythrocyte and anti-Forssman sera caused dissolu-
tion of cartilage matrix, when added to fetal chick bones in culture.
2. Only the cells a t the surface of the explants were killed by antisera,
whereas t he bulk
of
osteocytes and chondrocytes remained viable in the
absence of soIid cartilage matrix.
3.
Complement was necessary for the production of these changes by
both purified IgG and IgM fractions of antisera. Addition of C’6 to serum
deficient in this factor conferred the ability to activate antibody; the
activity of fresh se rum could b e abolished by absorption w ith antigen-
antibody complexes o r zymosan.
4.
Breakdown of cartilage matrix was associated with increased syn-
thesis a n d release into th e media of lysosomal acid prote ase iden tified
largely as cathepsin
D.
Antisera directed against the purified enzyme
prevented cartilage matrix breakdown induced by a variety of procedures
(Weston
et
al., 1969) .
5. The effects of complement-sufficient antisera upon cartilage matrix
and enzyme release may be overcome by cortisol (a stabilizer of Iyso-
somes; Weissmann an d Dingle, 19 61 ) or EACA ( a n inhibitor of t he acid
protease of cartilage; Ali, 196 4). These experiments suggest that T yp e
11
reactions result in the activation of complement at the cell membrane
and secondary events cause the lysosonial system to extrude its enzymes
into extracellular matrix. The subsequent hydrolysis by cathepsin D of
the major constituent of cartilage matrix (PP-L) leads to loss of meta-
chromasia. Two points warrant elaboration, however. First, whereas
EACA clearly inhibits crude acid protease activity of cartilage and the
breakdown of tissue (Fell et al., 1966) this inhibitor is ineffective upon
more purified cathepsin
D
( Rarrett, 19 69 ), ev en from liver. F urthermo re,
antisera directed against cartilage cathepsin D ( antigenically identical
to that of liver) do not entirely abolish cartilage breakdown, especially
over longer periods (W eston
et
a]., 1969 ). These points may indicate that
lysosomes contain othcr proteases, inhibited by EACA, which can de-
grade PP-L. Indeed, such activity has been identified in granulocyte
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
311
lysosomes that contain a neutral, EACA-inhibited protease active on
PP-L ( Weissmann and Spilberg, 1968).
Another Type
I1
reaction which has been extensively studied
is
nephrotoxic nephritis. The contributions of neutrophile lysosomes to the
lesions of this model glomerulitis were reviewed in detail by Cochrane
( 1968) . In brief, heterologous antibodies directed against glomerular
basement membrane become fixed to glomeruli, complement is bound
and activated, factors chemotactic for neutrophiles are elaborated, and
the white cells come into direct contact with the naked basement mem-
brane. The subsequent dissolution of the membrane can
be
monitored
by analysis for its degradation products in urine. Cochrane and Aikin
(1966) suggest that cathepsins
D
a nd
E
are responsible because the
capacity for degrading basement membrane of lysates prepared from
rabbit neutrophiles parallel their capacity to split denatured hemoglobin
at p H 2.5 ( p H optimum of cathepsin
E
= 2.5, of cathepsin D = 3.4) .
Janoff a n d Scherer (1 96 8) , studying lysates of
liuman
leukocyte lyso-
somes, have presented evidence that their elastolytic activity, at netural
pH, could account for hydrolysis of elastic fibers in h um an kidney in vitro
a nd dog
aorta
in vivo. The elastase has been partially purified and shown
capable of hydrolyzing benzyloxycarbonyl-L-alanine p-nitrophenyl ester-
a
synthetic elastase substrate a t near-neutral p H (Janoff, 196 9). Several
real problems remain unsolved in this model system. The neutrophiles
possess granule enzymes capable of hydrolyzing basement membranes,
and degraded fragments of the latter escape in the urine. Neutrophiles
are directly apposed to the basement membranes, but there has been
no suggestion as to how the enzymes might b e released. T he neutrophiles
are
not
degranulated, their membranes have not been shown to be
comprom ised, nor has direct exocytosis of granule co ntents been observed
(Cochrane , 19 70) . Fur ther work
on
this problem should do much for
our understanding of tissue injury, and an
in
vitro model utilizing micro-
pore filters impregnated with immune reactants has already shown that
neutrophiles release azurophile enzymes upon contact with the prepared
surface (Henson, 1970).
C. TYPE11 REACTIONS
The union of antigen with certain types of antibody results in the
formation of immune complexes, which in soluble or insoluble form, can
enter cc>llsby endocytosis. The major contributions to uptake of immune
complexes arc made by cells of the reticuloendothelial system and by
leukocytes (Fennel and Santaniaria, 1962; Dixon, 1963; Grant et al.,
196 7). Thereafter, the consequences
of
uptake to endocytic cells are due
to ( 1 ) enhanced cwdocytosis per se and ( 2 ) the na ture of the material
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312 GERALD WEISSMANN
AND
PETER
DUKOR
taken up , which, in t h e case of imm une complexes, includes com ponents
of the complement sequence ( Miiller-Eberhardt, 196 8). Daems an d Oo rt
(19 62) first showed th at imm une complexes we re taken u p by leukocyte
lysosomes, the ultrastructural appearance of which became altered in the
process. Thomas
(
1964) then demonstrated that isolated neutrophile
lysosomes could substitute for intact neutrophiles in reversed passive
Arthus reactions induced in leukopenic rabbits. One interpretation of
these experiments is that neutrophile granules, presumably taken up by
histiocytes, etc., supplied the missing inflammatory substances. Such
studies suggested that the uptake of immune complexes by lysosomes
containing neutrophilic materials was followed by inflammation in the
immediate vicinity of the endocytosing cell. This was not entirely
surprising, since Metchnikoff ( 1905) ha d first described this pheno men on:
“The leucocytes, having arrived at the spot where the intruders are
found, seize the m aft er the man ner of th e amoeba an d within their bodies
subject them to intracellular digestion. This digestion takes place in the
vacuoles in which usually is a weakly acid fluid which contains digestive
ferments (c yt as es ); of these, a very considerable nu m be r are now recog-
nized. T he cytases mu st be gro up ed with soluble ferments which a re
not thrown off by the phagocytes
so
long as these remain intact. Im-
mediately these cells are injured, however, they allow
a
part of their
cytases to escape.”
In a series of studies, Movat and collaborators (Lovett and Movat,
1966; Movat
et
al.,
1964,
1968; Taichman an d M ovat, 1966) have estab-
lished that immune complexes follow the route in phagocytic cells of
other particulates. They are endocytosed in phagosomes of leukocytes
and platelets. The phagosomes merge with primary lysosomes to form
secondary lysosomes. Consequently, an d by less well-studied mechanisms,
lysosomal enzymes are released extracellularly. Following immune com-
plex formation
in v i m ,
a t
a
time when uptake of microprecipitates of
antigen and antibody had been demonstrated by ultrastructural means,
levels of acid protease (de na tur ed hemoglobin, p H 2.5) , 0-glucuronidase,
and acid phosphatase became elevated significantly in the serum
(
Movat
e t
al.,
1968) . In vitro studies of passive cutaneous anaphylaxis in the
guinea pig showed that neutrophiles could endocytose microprecipitates
of antigen (ferri t in) and antibody (hyperim mu ne antiferri t in) into
secondary lysosomes. Later stages of particle uptake were associated with
“degranulation” of th e endocytic cell (L ov ett and Movat, 19 66 ). Fur ther-
more, it was found that substances released from leukocytes that had
ingested im mu ne complexes we re capable of inducing local capillary
permeability (Burke et al., 1964). By means of protease inhibitors, this
permeability factor could be distinguished from the basic, inflammatory
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THE ROLE OF LYSOSOMES IN IMMUNE
RESPONSES
313
proteins described by others (Janoff and Zweifach, 1964; Golub and
Spitznagel, 1966; Ranadive and Cochrane, 1968). Grant e t
al.
(1967)
foun d tha t union of antigen
(
ferritin) with antibody need not necessarily
proceed in the circulation or in vessel walls and have emphasized the
extravascular events which may be early triggers of the local Arthus
reaction. Astorga and Bollet (1965) showed that leukocytes could
endocytose complexes of rheumatoid factor with y-globulin; three lyso-
soma1 hydrolases becam e red istributed in these cells, presum ably d u e to
formation of more fragile, secondary lysosomes.
Not only neutrophiles are involved in particle uptake. Treadwell
(1965) has conducted several studies of the role of Kupffer cells in
systemic anaphylaxis in the mouse. Within
15
to
20
minutes after chal-
lenge of sensitized animals by antigen or complexes, plasma acid phos-
phatase showed six- to sevenfold increases; the liver content of the
enzyme was decreased concordantly. Further studies showed that these
changes were due to the disruption of Kupffer cells after these had
endocytosed immune precipitates
(
Santos-Buch and Treadwell, 1967).
Recently, Treadwell ( 1969) established that increments of lysosomal acid
phosp hatase in th e plasma-and decrem ents in enzym e content of th e
liver-could b e used as sensitive indexes of genetic susceptibility to an a-
phylaxis in some mouse strains (W-BR VS, S JL /J ) vs. others (C 57 B L/ 6J ).
Such studies suggest not only that immune complexes are taken up
by lysosomes bu t also th at th e enco unter of complexes with th e organelles
leads to escape into the circulation of previously sequestered enzymes
and biologically active materials. Possibly, under these abnormal situa-
tions
(
hyperimmunization, heterologous antibody )
,
cells are killed by
some other means and lysosomal hydrolases are released pari
passu
with
other cellular constituents. Since none of the above studies was monitored
with a ppr opria te enzyme markers for other organelles or the cell sa p
itself, we must turn to data on phagocytosis of particulates in general.
From data in Tables I11 and IV, it appears that polymorphonuclear
leukocytes extrude a portion of their hydrolases when forced to take up
zymosan (May et al., 1970). These observations are in accord with other
studies (Baehner et al., 1969; Pruzanski and Patterson, 1967; W. E.
Parish, 1969; Holmes et
al.,
1969; Crowder et al., 1969) on extrusion of
lysosomal hydrolases during particle uptake. These, too, are open to
criticism, since release of hydrolases after ingestion of inert particles can
b e explained on th e basis of diminished viability, nonspecific da m ag e to
the plasma membrane during endocytosis, contamination by endotoxin,
etc. In two of these studies (Crowder et ul., 1969; May et al., 1970) ,
viability was assayed by dye c,xclusion. Baehner et al. (1969) found that
a soluble cytoplasmic enzym e ( catalase ) was extruded from human poly-
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314 GERALD
WEXSSMANN AND
PETER DUKOR
morphs during uptake of starch particles, although to a lesser extent than
the concomitantIy extruded lysosomal hydrolases. In investigating this
problem, we have exposed mouse peritoneal macrophages to zymosan
(Dukor and Weissmann, 1970) and found no effect upon viability or
extrusion of lactate dehydrogenase ( a cytoplasmic enzyme) under cir-
cumstances in which considerable amounts
of
lysosomal @-glucuronidase
were released (Table V ) . Release of lysosomal hydrolases, at least with
zymosan,
is not
associated with nonspecific release of LDH, although
dead cells readily release both enzymes. Furthermore, it
is
also possible
to block release of @-glucuronidase from macrophages by addition of
cyclic AMP. The latter point needs to be explained. The work of Lichten-
stein and Margolis (1968), Levy and Carlton (1969), and Malawista
(1968) suggests that two types of agents can block release of histamine
from blood basophiles by antigen. One group (cyclic AMP, methyl
xanthines) apparently acts by raising the intracellular level of cyclic
AMP; whereas, the other group includes modalities (colchicine, cold
temperatures, D,O ) that interfere with the function or aggregation of
microtubules. Now the relationship that these two groups bear to each
other is by no means clear, but it has become common to attribute to
microtubules a function in the directed flow and merger of cytoplasmic
organelles (Malawista, 1965; Porter, 1966; Lacy
et
al.,
1968;
R.
Hirsch-
horn et al., 1970a). It may well be, therefore, that microtubules regulate
TABLE
V
ENZYMEELEASE Y ZYMOSANORFREEZINGN D
THAWING)
ROM
CULTURED OUSEPERITONEALACROPHAGEP
~~ ~
’ Cells with 8-Glucuroni- Lactic dehy- Cells
Culture conditions particles ( ) b released ( )c eosin Y
ingest>ed dase released drogenase excluding
Controls -
1 .0
f 0 . 3
0 . 2
0.5 99.2 f
0 .4
Zymosane
83.2 rt
5 . 7 8 . 9
k
0 . 4 0 . 1
f 0 . 3
98.5
f . 9
Zymosan and adenosine
3’,5’-
88.3 r t
2 . 0
3 . 9 f 0 . 3
ND
9 8 . 7
f
0 . 6
Freezing and thawingd
-
60.3 f
. 9 38.9 f
6 . 4
0
monophosphate
lop3
M
~~
a Thioglycollate-induced peritoneal mononuclear phagocytes cultured for 24 hours
in medium “199” containing 10% fetal calf serum (2
x
108 cells in 2 ml. per culture).
Particle uptake, viability, and enzyme release determined after a further 2-hour period;
6-12 cultures per group.
b Activity of supernatant expressed in per cent of total enzyme activity of culture
after six cycles of freezing and thawing.
c
Activity of supernatant expressed in per cent of total enzyme activity of culture
after additinn of
0.1%
T r h n
X-100.
e
Particles per culture,
4 X lo7 .
One cycle a t the beginning of the 2-hour period.
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
315
the flow of phagosomes to primary lysosomes, a site sensitive to coIchi-
cine, as Malawista and Bcnsch (1967) suggest.
If
this sitc were also
sensitive to th e intrace llular level of cyclic nucleotides, th e release or
extrusion of hydrolases from nentrophiles might be modified by those
agents that, in basophiles, modify release of histamine. This discussion
has focused upon the zyinosan model, but since immune complexes
provoke the release of hydrolases from intact cells (a point that deserves
still further critical evaluation), the situation should hold for uptake of
antigenantibody part iculates .
There is no question (C och rane , 1968) that immune complexes can
generate chemotactic factors from complement components in the fluid
phase. Thus they call forth the cellular machinery for their own disposal
by the endocytic process. But do components of the complement system
play a role in subsequent events within the endocytic cell or its environ-
ment? Is the system capab le of self-amplification? Following experiments
in which leukocytes ( Hurley, 1964) or their lysosomes (Cornely, 1966;
Bore1 et al., 1969) were shown to generate chemotactic factors from
fresh serum, W ar d a nd Hill (1 97 0) identified a neutral protease in rab bit
leukocyte lysosomes. The enzyme was shown capable of generating
chemotactic activity by means of a complement component. Indeed,
lysates of the granules cleaved
C 5
t o
C5a
fragments which varied in
molecular weight from 4 to 15,000. The enzyme, which had a pH
optimum of
7.2
to
7 .3 ,
was inhibited by soybean trypsin inhibitor, EACA,
TAME, and BAA methyl ester. Hill and Ward (1969) had previously
shown that a neutral protease from heart t issue could generate chemo-
tactic activity from C’3; this protcase was also inhibited by trypsin
inhibitor, TAME, and BAA. Taubinan et al. (1970) have recently con-
firmed the generation of chemotactic activity from C’5 and, furthermore,
demonstrated that neutrophile lysosomes can cleave C’3 into large and
small fragments-as yet not biologically active. Additiona lly, lysosomal
fractions can both activate C’ls to C’lS, and inactivate C’lS. Such studies
indica te th at lysosomal enzymes-in th e absence of imm une reactions-
can induce alterations usually associated with sequential, immunological
activation of the coniplement system. It is thus likely that when leuko-
cytes in plasma endocytose inert particles or immune precipitates thcy
may also take up components of th e com plemen t system in various stages
of
activation. Recent studies with m odel membranes, liposomes (reviewed
by Sessa and Weissmann, 19 68 ), have shown th at th e activated coniple-
ment sequence C’l
+
C’9 can disrupt these lipid models for lysosomes or
other biomembranes (Haxby et al., 1969) and tha t
C’5-9,
acting via a
phospholipase
C
disrupts liposomes (Lachmann et al., 1970).
It is
of
interest, therefore, to determine whether
one
or another of the lipid-
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316
GERALD WEISSMANN
AND
PETER DUKOR
disruptive components ( C'8, C'9), activated inside secondary lysosomes
during endocytosis, is involved in the damage to cells following uptake
of immune complexes.
One further mechanism for tissue injury in immune complex disease
must be mentioned. When leukocytes are injured in Arthus reactions,
some or many of their lysosomal granules are extruded in intact form
( Lovett and Movat, 1966). Now, when leukocytes are exposed to particles
such as latex or zymosan, they can take up many of these without a major
loss of viability. But when neutrophiles were permitted to ingest isolated
(heterologous) leukocyte granules, they underwent prompt and rapid
cell lysis (Wiederman et at., 1966; R. Hirschhorn and Weissmann, 1967).
This was not brought about when cells were exposed to lysates of the
granules and, presumably, was due to the activation of one or another
granule product during ingestion. Macrophages do not undergo destruc-
tion after uptake of neutrophile granules. It remains
to
be determined
whether isologous granules will induce similar degrees of cell death.
D. TYPEV REACTIONS
Lymphocytes, presumably derived from the thymus (Mitchison,
1969b)
,
undergo transformation into cells capable of inflicting tissue
injury after their encounter
in uitro
and ? in vivo with ( I ) antigens to
which the donor has become sensitized (Pearmain
et al.,
1963)
( 2 )
allogeneic cells (Dutton, 1965) 3 ) nonspecific mitogens from
Phuseolus
vulgaris (PHA) (Nowell, 1960), or PWM (Farnes et
aE.,
1964),
( 4 )
antisera against allotoypic determinants of immunoglobulins
(
Sell and
Gell, 1965), and ( 5 ) antisera against surface antigens of the lymphocytes
themselves (Grasbeck
et
al., 1963). The transformed lymphocyte
in
culture has been shown to elaborate several biologically active factors
into the medium. These include a macrophage inhibitory factor (David,
1966), a factor capable of inducing transformation and mitosis of resting
lymphocytes (Maini e t
al.,
1969), a factor capable of inducing local
inflammation
(B .
Bennett and Bloom, 1968), a transfer factor (Lawrence,
1969), a factor capable of inducing responsiveness to specific antigens in
unsensitized lymphocytes ( Valentine and Lawrence, 1969), a factor
chemotactic for other macrophages (Ward
et al.,
1969), and a factor
which is cytotoxic to target cells (Granger and Kolb, 1968). This subject
has been reviewed extensively in a recent volume (Lawrence and Landy,
1969). The relationship of these factors to lysosomes and the vacuolar
system is by no means established, but each of them is released at a
time when the vacuolar system is activated. The transformed lympho-
cyte is rich in lysosomes (Allison and Malucci, 1964; Parker
et al.,
1965;
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THE ROLE OF LYSOSOMES IN IMMUNE RESPONSES
317
R. Hirschhorn et al., 196 5), whercas there is a paucity of these organelles
in normal or resting blood lymphocytes. Within 36 to 48 hours after cells
are stiinulated by PHA or antigen, new, acid phosphatase-positive
granules ap pp ear . T h e cells’ total contcnt of acid @ -glycerolphosphatase,
acid phenolphthalein phosphatase, and aryl sulfatase undergoes signifi-
can t increases (R . Hirschhorn et al., 196 7). I t m ust be pointed out , how-
ever, that other organelles (Douglas et
al.,
1967; Ch apm an d
al.,
1967;
Ha lpeni et al.,
1968;
Clausen and Bouroncle, 1969) such as the endo-
plasmic reticulum an d the Golgi app aratus (a n d their associated en-
zymes ), undergo similar development. Histochemical studies of trans-
formed lymphoid cells engaged in the destruction of target tissues have
shown that the invading lymphocyte
is
rich
i n
a variety
of
primary and
secondary Iysosomes; indeed, the latter show residual evidence of earlier
endocytosis (Weiss, 1968; Brandes et al., 19 69). Resting lymphocytes do
not attach to target cells and are poorly phagocytic (Robineaux et
a,?.,
19 69 ). In co ntrast, th e plasma m emb ranes of transform ed cells are
closely opp osed to ( Weiss, 196 8) or even joined with ( Brandes e t al., 1969)
those of target cells. Uptake of materials such as endotoxin, immuno-
globulins, peroxidase, or neutral red is considerably enhanced in the
transformed cell (Hirschhorn et al., 1968; Robineaux et al., 1969) . In
oitro transformation of lymphocytes is followed by mitosis, and many
investigators have presented evidence that lysosomes of lymphocytes
and other cells (Robbins and Gonatas, 1964; Allison and Malucci, 1964;
R. Hirschhorn et
al.,
1965; Kent et
al.,
1965; Bastos e t al., 1967) undergo
morphological changes at mitosis. Perhaps their hydrolases are required
for the remodelling of cells du rin g division ( R . Hirschhorn et al.,
1965;
Allison, 19 69 ). Critica l evidence to this poin t is still lacking.
Lysosomes and the vacuolar system are clearly invoIved early in the
course of lymphocyte transformation. Allison and Malucci
(
1964) de-
scribed that, within a few hours after PHA stimulation of lymphocytes,
there was an increased p ermeability of their m embranes to th e sub strate
for acid phosphatase. In a detailed biochemical study of these events,
Brittinger et at. (1968) and R. Hirschhorn e t al. (1968) established that
within 2 to 4 hours after stimu lation of lym phocy tes by P HA , lysosomal
hydrolases
(
but not malate dehydrogenase ) become redistributed from
granular to less-sedimentable fractions of cell hom ogenates (T a b le VI )
.
These changes are accom panied by enh ance d permeability of lymphocyte
membranes to substances
in
the ambient medium such as neutral red.
Furthermore, Brittinger e t
al.
(1969) were able to demonstrate that a
nonaggregating mitogen ( PWM ) also induced redistribution of lysosomal
hydrolases. Other studies had shown that changes in the nuclei of trans-
formed cells [acetylation of histones (Pogo et al., 1966), turnover
of
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TABLE
V I
DISTRIBUTIONF ENZYMESMONG SUBCELLULAR
RACTIONS
ERIVEDROM LY
I N C U B A T E D
WITH
AND
WITHOUT PHYTOHEMAGGLUTININ-P FOR
120 hfINUT
No. of
Fractiona
Treat- experi-
Enzyme mentb ments Debris Nuclei Granules
P f
SED
8-Glucuronidase Cont,rol
Acid phosphat.ase Control
Malate dehydrogenase Control
Protein Control
PHA-P
PHA-P
PHA-P
PHA-P
13 5.6
f 1 . 0
13.2
f 1.1
69.3
f
1 .4
<0.001
13 3.6
f
0 .5 18.7 2 2 . 8
57.8 f 2 . 3 + 2.4 5
8
4 .9
f
1.6 15.7
k
2 . 0 61.6 k 2 . 7
<0.05
8
3 .7 f .8
18.5 f . 3
53.9 f 2 . 4 ic3.21
5 3.0
f
2.2 13.0
k
. 2 27 .5 1 . 9 Not signific
5
3 . 5
k
0 .9 1 6. 3 k 4 . 0 3 1. 3
k
2 . 9 f 3 . 44
10 9.6 f . 7 10 . 0 k 1 .2 16 .6
k
1 . 7 Not signific
10 7.2
f
1 . 4 11 . 5
4
1 .5
16.2
+
0 . 9 51 . 80
a
Values are given
as
per cent
of
the total recovered activity i SEM.
PHA-P = phytohemagglutinin-P.
Paired t-test
i
standard error
of
differences between paired samples (Hirschhorn et
al.,
1968)
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THE
ROLE OF LYSOSOMES
IN IMMUNE
RESPONSES
319
phosphoproteins ( Kleinsniith e t
al . ,
l966), and ei ihanced binding of
actinomycin D (Darzynkiewicz et uZ.,
1969)
or acridine orange (Kil-
lan de r an d Rigler, 19 69 ) might indicate tha t controls of th e transcription
of DNA were affected by PHA. One possible sequence of events, there-
fore, app eare d am enab le to fu rther study. T he encounter of resting
lymphocytes with PHA (o r antigens in th e case
of
sensitized cells) might
lead to enhanced endocytic activiy ( R . Hirschhorn e t al . , 1 x 8 ) . T he
redistribution of lysosomal hydrolases which accompanied these events
might render available to the resting nucleus a neutral protease that
would remove repressor proteins (?histones) from DNA, making pre-
Template ac t iv i t y
of
l ymphocyte nuc le i
for bacter ial RNA-polymerase
(control vs P H A )
a
z
r
5
c
m
E
a
a
500I
I I
1500
-
1000
-
-
0
10 20
30
40
0
:
pg.
Nuclear DNA
FIG. 2. Effect of stimulation of lympliocytcs with phytoheniagglutinin ( PHA
)
for 4 hours on template capacity of the isolated nuclei. Lymphocytes were incubated
for 4 hours with 0 ) nd without 0)HA; nuclei were isolated and incubated for
10 min iits at 37°C. in 0.5 ml. containing 58 pnioles Tris buffer ( p H 7.5) , 1.2 pmoles
spermidine HCl, 1.17 fimoles MnSO,, 1 pniole MgC12, 0.450 pmole each of the four
nucleoside triphosphates, 3.33 pcuries 'HH-guanosine triphosphate CTP )
(
-*3
iiipmoles), 2-50 pg. nuclear DNA, and 50 units RNA polymerase iVicrococcils
Zyso-
deikticiu, Miles Laboratories ) ; and '€1-GTP incorporation was determined. Each value
is the average of duplicate determinations of counts per minute ( c.p.m. incorporated
in the absence of actinomycin D minus the counts per miniite incorporated
in
the
presence of actinoiiiycin
D
( R . Hirschhorn et
al.,
1969).
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320
GERALD WEISSMANN AND PETER DUKOR
viously repressed sites available for transcription by RNA polymerase.
Histochem ical evidence for dissociation of DN A from histones has, in
fact, been obtained by Zetterberg and Auer (1969) and Rigler and
Killander (1 96 9) . A furthe r precedent for this sequence can be add uce d
from the studies of Allfrey et
al.
(19 63) , who found that t reatment with
trypsin augmented the template capacity of nuclei for endogenous RNA
polymerase.
To
test this hypothesis, nuclei were isolated from cells
4
hours after PHA stimulation an d from controls, a nd t h e tem pla te capacity
of th e former was foun d considerably enhanced (F ig .
2 ) ( R .
Hirschhorn
et al., 19 69 ). Furtherm ore, experiments with frozen-thawed an d deter-
gent-treated nuclei excluded the possibility that changes in the nuclear
membrane accounted for the augmentation of template activity. Perhaps
most persuasive were experiments in which trypsin was added to nuclei
from control and stimulated cells. Although the enzyme clearly aug-
mented the template capacity of both nuclear preparations (Table VII ) ,
the relative enhancement of template activity by trypsin was clearly
diminished in nuclei from PHA-stimulated cells. One explanation for
these findings, in line with the above hypothesis, was that a neutral
TABLE VII
EFFECTOF
TRYPSINN THE TEMPLATE
APACITYF
NUCLEI
SOLATED
F R O M
PHYTOHEMAGGLUTININ-STIMULATED
AND NONSTIMULATEDUMAN YMPHOCYTES&
No. of
Trypsiii experi-
(pg./ml.) ments
0 9
1.25 2
10.7 3
12.5 2
25.0 2
PHA Coiitrol
1,370 f 225
10,050 k 550
14,100
f
61
13,250 f 50
25,500 k ,500
370 k 24
5,320 f 43
8,880
f
156
10,450 f 1,909
12,200
k
1,300
Average with trypsin
P I C ratio
3.56 f .507b
1 .89 f 0.071
1 . 6 0
2
0.102
1.29 k 0.150
2 . 0 8
f
.143
1 . 7 k 0.11OC
a Nuclei isolated from cells cultured with and without phytohemagglutinin (PHA)
for 2 hours were incubated for 20 minutes at 37°C. with trypsin (1.25-25 pg./ml.) and
the reaction stopped by addition of soybean trypsin inhibitor
(2..5-50
pg./ml.). The
capacity
of treated nuclei to prime
for
incorporation of 3€I-gnanosine riphosphate (GTP)
into RNA in the presence
of
exogenous RNA polymerase was determined. Values repre-
sent actinomycin D-sensitive (c.p.m.) 3H-GTP incorporated/100 pg. DNA
f EM.
The amount of DNA per assay was varied between
10
and
20
pg .
for trypsin-treated
nuclei and between
10
and
40
pg. for nontrypsin-treated nuclei. P I C ratio
=
incorpora-
tion 3H-GTP into RNA by nuclei
from
PHA-treated cells/incorporation of 3H-GTI' into
RNA by nuclei from untreated cells.
b P <
0.005
(paired &test) PHA versus control.
P < 0.005 (t-test) PIC ratio without trypsin versus trypain average (It.Hirschhorn
et al.,
1969).
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THE
ROLE OF
LYSOSOMES
IN IMMUNE RESPONSES
321
protease had preemptively acted upon protease-sensitive sites in stimu-
lated cells. Inde ed, Uyeki and Llacer (19 68) were a ble to enhance DNA
synthesis of spleen cell culturcs by cxposing them to trypsin. To test
fu rth er th c general hypothesis, inhibitors of p rotease action were ad de d
to cells exposed to PHA, and it was found that EACA, TAME, TLCK,
an d TPCK inh ibited the expected response of lym phocytes to the m itogen
( R. Hirschhorn et al. , 19 70 b). Furthermore, in experiments analogous to
those discussed above with neutrophiles and macrophages
( R .
Hirsch-
horn et al. , 197 0a), i t was found that cyclic AMP ( M ) , its di-
butyryl derivative ( >1W M ) , and theophylline ( > M ) , each di-
minished the response of celIs (D N A , RNA synthesis) to PHA. When
lower concentrations of cyclic AMP (between
3.3
x
1W
to
3.3
X
10
M )
were added to resting lymphocytes, augmented incorporation of tritiated
thymidine was observed. These biphasic responses suggest that protein
kinase may be the crucial enzyme involved (Miyamoto et al., 1969) ; the
enzyme is activated by low, and inhibited by high, concentrations of
cyclic AMP. Since the transformation process is sensitive to variations
in the level within cells of cyclic AMP, the hypothesis is reinforced that
the merger and flow of components of the vacuolar system display
similar sensitivity.
Re causr som e of the agents that nonspecifically stimu late lympho-
cytes also disrupt isolated lysosomes, e.g., streptolysin S, staphylococcal
a-toxin, Hg”, ultraviolet irradiation, and because some
of
the agents
that stabilize lysosomes also inhibit lymphocyte transformation, e.g.,
cortisone or chloroquine, it was suggested tentatively that release of
liydrolases from lysosomes within cells initiates lymphocyte transforma-
tion ( K. Hirschho rn and Hirschhorn, 1 96 5) . Reevaluation of these data ,
together with the recent demonstration that the lysosome-disruptive
factor in streptolysin S could be dissociated from its mitogenic activity
(Taran ta et ( i l . , 1969) suggest that this concept is no longer tenable in
its original form. By virtue of the possible role of the adenyl cyclase
system (s ee ab ov e) a nd th e inhibition by ouabain of lymphocyte trans-
formation (Quastel et al. , 1969), it now becomes likely that nonspecific
mitogens i n all ceZls, and antigens in some cells, perturb the surface of
the cell. Most agents active on lysosomes in vitro have similar effects at
the plasma mem brane (revie w by Weissniann, 19 69). Surface m em bran e
alterations, by means as yet unexplored, subsequently induce rearrange-
men t of intracellular vacuoles. Fisher and M ueller (1 96 8) ha ve indeed
shown increm ents of th e incorporation of 32 P0 ,2 - into pho sphatidyl
inositol shortly after PHA. Critical experiments are still lacking, however,
to test th e hypothesis t ha t lysosomal proteases ar e responsible for changes
in the nucleus after th e primary stimulus to th e vacuolar system has acted
at the cell periphery.
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322
GERALD WEISSMANN AND PETER DUKOR
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Molecular Size and Conformation
of
Immunoglobulins
KEITH
J. DORRINGTON',' AND CHARLES TANFORD
M.
R.
C . Mo l e cu l a r P h a r m a co l o g y U n i f, Me d i c a l Sch o o l , U n ive r s it y
of
C a m b r i d g e ,
C a m b r i d g e , E n g l an d , a n d D e p ar t m e n t o f Biochemist ry, Duke
U ni ver s it y Me d i c a l C e n t e r , D u r h a m, N o r t h C a r o l i n a
I. Introduction . . . . . . . . . . .
11.
Molecular Size of Immunoglobulins and Subunits
. . .
A.
yG-Globulins
. . . . . . . . . .
B. yM-Globulins . . . . . . . . . .
D. Other Immunoglobulins
. . . . . . . .
111. Conformation of I~nn~unoglobulinsnd Subunits . . .
A. Overall Shape and Flexibility
. . . . . .
B . Internal Folding . . . . . . . . .
C. Properties of Separated Heavy and Light Chains .
.
Recovery of Native Conforniation Following Chain
Dissociation and Unfolding
. . . . . . . .
A. Reversible Random-Coil Formation
. . . . .
B. Reversible Dissociation into Half-Molecules
. . .
C. Dissociation into Heavy and Light Chains and Its Reversal
V.
Conclusions . . . . . . . . . . .
References
. . . . . . . . . . .
C. yA-Globulins . . . . . . . . . .
IV.
. 333
. 334
. 334
. 337
. 338
.
340
. 341
. 341
. 356
. 363
. 366
. 366
. 370
.
371
. 375
.
376
I. Introduction
One of the most exciting problems in molecular biology today is the
elucidation of the relationship between the biological properties of im-
munoglobulins and their organization at the several levels of protein
structure.
Significant advances have been m ade in th e determination of th e amino
acid sequence of immunoglobulin subunits, particularly from myeloma
proteins. T h e results of sequence studies have been extensively reviewed
with particular reference to the genetic and evolutionary information
they are thought to convey (Len nox and Cohn, 1967; Cohen an d M ilstein,
1967; Ed elm an a nd G all, 196 9). I t is not our intention to reiterate these
findings in detail except where they have yielded information on the
particular stru ctu ral feature u nder discussion. Th e present review is
primarily concerned with studies on the higher levels of organization in
immunoglobulin molecules; their size and shape as well as the more
intimate aspects of their internal folding.
tenure of
a
Wellcome Travelling Fellowship,
1966
and
1967,
at Duke University.
Canada.
' A
portion of the work described in this review was carried out during the
*
Perinanent address: Department of Biochemistry, University of Toronto, Toronto,
333
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334
KEITH J.
DORRINGTON AND
CHARLES TANFORD
I I
Mo lecu la r S i ze of I mmuno g lo bu l ins
a n d
Subunit s
T h e molecular size of the various immunoglobulins and their subunits
are discussed separately in this section. However, to aid comparisons
some of the most definitive data (in our opinion) are collected together
in Table I.
T A B L E
I
MOLECULAR
EIGHTS
O F
I M M U N O G L O B U L I N S
AN11 T H E I R POLYPEPTIDE
C H A I N S a
Immnnoglob- M ~ I . t.
x 10-3
di n or
polypeptide Polypeptide
chain Species studied Total por'lionb
TG-Globulin
7 Chain
Light chainc
?M-Globulin
yM, Subunit
p
Chain
I.L Chain
7
A-Globdin
al
Chain
aZChain
?A-Globulin
a
Chain
7D-Globulin
S
Chain
e
Chain
-yE-Globdin
Human, rabbit, horse
Human, rabbit, horse
Human, rabbit, horse
Human, rabbit,, shark, lamprey
Human, rabbit
Human, rabbit
Shark, lamprey
Human serum, 7
S
Human serum, 9-10 S
Human, rabbit secretory
Human
Htiman
Mouse
Mouse
Human
Human
Human
Human
143-149
52-54
22-23.5
800-950
175-185
65-70
70-77
158-160
318
56-58
S2-53
115-120
<50
175- 180
60-66
185- 190
71-73
370-390
These figures reflect the authors' prejudices regarding the reliability of different
molecular weight determinations. See test for full listing of results.
b The mass
of
the carbohydrate has been subtracted from the molecular weights of
all heavy chains
t o
facilitate comparison
of
t,he lengths
of
the polypeptide chains per
se.
c All available evidence indicates that the same light chains are utilized in the as-
sembly of all immunoglobnliils, regardless of heavy-chain category, and that the different
classes
of
light chain have nearly the same inolecular weight.
d Level of carbohydrate has not been det,ermined.
A.
yG-GLOBULINS
Estimates of the molecular weight of yG-globulins prior
to
1960 have
been tabulated by Porter (19 60 ). Th e values obtained ranged from
150,000
to 190,000 an d, in general, were ab ove those ob tained more re -
cently (
see
below ) . Ultracentrifugal studies provided most of the
early data although osinotic pressure, light scattering, and low-angle
X-ray scattering were also used.
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MOLECULAR
SIZE
AND CONFORMATION OF IMMUNOGLOBULINS 335
In recent years attention to the concentration dep end ence of th e
molecular weight values an d th e effects of aggregates in the yG pr ep ara -
tions has resulted in lower values for the molecular weight. Cammack
(19 62) obtained a value of 137,000 fo r rabb it yG using sedimentation
an d diffusion, taking into account the concentration dep end ence of the
parameters used. Failure to do this resulted in higher values, e.g.,
179,000 at 10 mg ./ml. T he sam e sample examined by th e Archibald
approach to equilibrium m ethod gave a value of 172,000 at 9.2 mg ./ml.
T h e effe ct tha t small amounts of polymerized yG can have on m olecular
weight values has been well illustrated by Pain (1965). A sample of
equine yG, apparently free of aggregates, gave a value of 151,000 by
sedimentation an d diffusion analysis. How ever, sedime ntation equ ilibrium
studies gave a weight-average molecular weight
( M , )
of 160,000 an d
a x-average value ( M , ) of n ea r 170,000 indica ting m ass hetero geneity.
Ge l filtration on Sep hadex G-200 eliminated trac e am ounts of p olymer
and the
M ,
dropped to 151,000. Similarly, rabbit yG gave a value of M,
near 141,000 imm ediately following gel filtration, but after concentration
an d storage for a f ew days th e value h ad increased to 157,000, presum-
ably owing to aggregation.
The development of the high-speed sedimentation equilibrium method
of Yphantis ( 1964) has resulted in more consistent and reproducible
molecular weights, at least for yG preparations. The use of low protein
concentrations in this method (
0.1-1.0
mg./ml . ) largely eliminates the
concentration dep end ence problem. Marler e t al. (1964) obtained a
value of 145,000 for rab bit yG from sed imentation equilibrium measure-
ments. A similar value (151,000) on the same sample was obtained from
sedimentation and viscosity data, using the equation of Scheraga and
Mandelkern ( 1953) (Noelken et al., 196 5). Small
and
L a mm ( 1966 ) ob-
tained a value of 140,000 for rab bit yG in 5.0 M guanidine hydrochloride
by sedimentation equilibrium. Equilibrium sedimentation studies by one
of us
(K.J.D.)
on human normal and myeloma yG-globulins have
yielded values fr om 143,000 to 148,000 bo th in d ilute salt solution a n d
6.0 M guanidine hydrochloride.
T h e problem s of aggregation a nd limited solubility of th e heavy chain
of yG-globulin (and other imrnunoglobulin classes) in neutral aqueous
solvents have meant that reliable values for their molecular size have
only been obtained in dissociating solvents. In p ractice this has also bee n
true for the light chain. Marler
et
al. (1964) deduced the molecular
weights of the chains of rabbit yG without prior separation. The yG was
reduced with 0.1 M 2-mercaptoethanol in 6.0 M guanidine hydrochloride
and subjected to equilibrium sedimentation at various protein concen-
trations in the same solvent. Values of number, weight, and z-average
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336
KEITH J. DORRINGTON AND CHARLES TANFORD
molecular weights were obtained and compared with similar values
for calculated equ imo lar mixtures of ch ains of assumed sizes. T he
results were compatible only with a four-chain model; the heavy chains
having a molecular size between 50,000 and 55,000 and the light chains
between 20,000 a nd 25,000. This conclusion was further tested by
calculating the solute distribution in the centrifugal
field
for equimolar
mixtures of th e chains assuming th e above molecular weight values a nd
comparing it with the act ua l distribution-within the experimental error
the distributions were identical. The molecular sizes for heavy and light
chains deduced by Marler et
al.
were in close agreement with those
of Pain (1963) an d Small et
al.
( 1963) who used the more conventional
approach of separating the chains prior to the molecular weight de-
terminations. A more precise study has been reported more recently by
Small and Lamm (1966) on rabbit yG chains. They se para ted the heavy
and light chains from extensively reduced and alkylated
yG
on Sephadex
G-200 in 5.0 M guanidine hydrochloride. The yields of heavy and light
chains were 68 and 32%, espectively, consistent with a four-chain model.
The rationalized “best” values for the molecular weights of the heavy
and light chains were 53,000 a nd 22,000 in
a
yG molecule of 140,000
molecular size. Some evidence was presented suggesting that complete
reduction of the
yG
was not achieved. However, the yields of heavy and
light chains appeared to be independent of the method used for reduc-
tion, so that the molecular weight values are probably unaffected.
Equine yG can be resolved into three antigenically distinct com-
ponents (Rockey e t al.,
1964).
The molecular size of the polypeptide
chains of yG,,, have been determined by Montgomery et al. (1969) .
Sedimentation equilibrium in 6.0 M guanidine hydrochloride and gel
filtration on calibrated columns of G-200 in 8.0
M
urea-0.05
M
propionic
acid gave values between 52,300 and 53,900 for the heavy chain and
22,300 and 23,100 for th e light chain.
Com men t on th e values of th e partial specific volume V ) chosen for
th e protein un der investigation might b e ap pro pria te here. Sedimenta-
tion eq uilibrium provides estimates of M ( 1 - p ) , where p is the solvent
density. T he final accuracy of
M
depend s on th e precision of th e V value
used; a 1 rror in V gives a 5 6 % ncertainty in M in guanidine or 3%
uncertainty in dilute aqueous salt solution. Although direct measure-
ments of D can be easily made pycnometrically in the relevant solvent,
large amounts of protein (250 mg. ) are required compared to the amount
needed for th e actu al molecular w eight determination (ca. 1 mg. ) . De-
termination of V with less protein requires sophisticated apparatus. For
proteins in dilute salt solution (ca. 0.1 M ) , a reasonable estimate of 5
can
be
obtained from the V’s of the constituent amino acids if an amino
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MOLECULAR SIZE
A N D
CONFORMATION OF IMMUNOGLOBULINS 337
acid analysis is available (C oh n and Edsall, 194 3). How ever, for glyco-
proteins ( e.g., immunoglobulins
)
some estimate of the contribution of
the carbohydrate
to
the overall
V
must be made in view
of
their low
V
values (ca. 0.6-0.65 ml. gm.-l) compared to amino acids. It is necessary
for the subunit molecular weight determinations in the presence of
guanidine hydrochloride or urea
to
estimate
a
value
of
the “effective
specific volume” of th e anh ydrous protein in the mixed solvent, This
quantity includes the actual V of the protein plus a correction term which
depends on the interaction of the protein with the solvent components
( Had e and Tanford, 19 67) .
As
discussed by a number
of
workers (Hade
and Tanford, 1967; Schachman and Edelstein, 1966) the preferential in-
teraction of protein with water (pr efe ren tial hy dra tion ) would lead
to
a
value for t he “effective V” larger than the V of the protein in dilute salt
solution. On the other hand, preferential guanidination would cause
little change and the effective V would closely approximate the V of the
native protein in dilute salt . Hade and Tanford (1967) have demon-
strated preferential guanidination for several proteins, and a number of
workers have shown that the effective V for anhydrous, salt free
yG-
globulin in concentrated guanidine hydrochloride, determined according
to the method of Casassa and Eisenberg (1961), is only slightly lower
than the corresponding quantity in dilute salt solutions (Marler
et
at.,
1964; Small and Lamm, 1966). The problems associated with molecular
weight determinations in three-component systems have recently been
discussed in thermodynamic terms by Reisler and Eisenberg ( 1969) .
W ith th e determination of the com plete sequence
of
the heavy chain
of one yG molecule (Edelman e t
al.,
19 69 ) we can calculate its molecular
weight to
be
48,600 (n o t including carbohyd rate w hich would bring it
up to 50,100) without the ambiguities due to uncertainties in V. T he
light chain
of
this protein
has a
molecular weight of 23,400, and the
molecular weight of t he whole m olecule is 144,000 (po lyp ep tide por-
t ion o nly) or 147,000 (carbohy drate inclu ded ).
B. yM-GLOBULINS
The YM-globulins are extensively reviewed by Metzger in a separate
chapter of this volume, and the discussion here will therefore
be
con-
fined to new molecular weight da ta by Do rrington a nd Mihaesco (19 70) .
These workers have determined th e molecular size of t w o intact Wald en-
strom macroglobulins together with their p chains and the various
fragments produced by papain and pepsin. Sedimentation equilibrium
(Yphantis, 19 64 ) of intact yM in dilute salt a nd in 6
M
guanidine hydro-
chloride yielded a value of 891,000 (S.D.
+20,000).
Provided care
was
taken to remove any polvmers
of
the 19
S
species, th e ob served molecular
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338
KEITH
J .
DORRINGTON
AND
CHARLES T.4NFORD
weight was insensitive to changes in rotor speed and initial protein con-
ccntration. The
p
chains were prepared from mildly reduced and alkyl-
ated yM and subsequently fully reduced in
6
M
guanidine hydrochloride
and purified by gel filtration. The molecular weight was found to be
65,200 ( S.D. *1800) ind ep en de nt of initial protein concentration a nd
rotor speed, using values of the partial specific volume calculated from
the amino acid and carbohydrate compositions. In addition, it has been
possible to determine the molecular size and relative location of regions
of the ,U chain corresponding to distinct antigenic determinants from the
molecular size of p-chain fragments within various proteolytic fragments.
A fuller discussion of these data will be deferred until Section 111.
It
seems established from th e above a n d othcr d ata (s ee Metzger, this
volume) that the molecular size of the
p
chain is greater than the y chain
of yG. This can only be partially accounted for by the higher carbo-
hydrate content of yM (7-11%) compared to yG ( 2 3 % ) .For the yM-
globulins referred t o above, of th e total mass of 65,200 for the p chain,
some 9000 gm./mole was due to carbohydrate leaving a polypeptide
portion of 56,200. This com pares with nearly 48,600 for th e polyp eptide
of the
y
chain (Edelman et nl., 19 69 ). It w ould seem, therefore, that
the chain is some 65 to 70 residues longer tha n th e
y
chain. Since
there is some evidence that the
p
chain appeared before the
y
chain in
evolution, the apparent deletion in y chain com pared to chain has
stimulated some interesting discussion (Hill e t al., 1967; Lennox and
Cohn, 1967).
C.
yA-GLOBULINS
The yA-globulins were reiewed by Tomasi (1968) in Vol. 9
of
this
series, and only the recent work on the molecular size will be discussed.
The molecular size of several monomeric and dimeric yA-globulins
have been determined by Dorrington and Rockey (19 70 a). All th e pro-
teins studied were isolated to a high degree of immuiiochemical purity.
The monomeric yA-globulins had sedimentation rates (s & , ~ ) etween
6.5 S and 6.7
S
and the dimeric yA, 8.6 S. The molecular weights of the
yA monomers w ere b etween 158,000 an d 160,000 as determined by
sedimentation equilibrium in either dilute salt or 6.0 M guanidine hydro-
chloride solutions. The yA dimer had
a
molecular weight
of
318,000
(S.D. -t7500) close to the value expected for a dimer of a 160,000
basic unit. The molecular size originally determined in dilute salt solu-
tion
was
unaffected by the presence of 6.0 M guanidine hydrochloride,
clearly indicating that the monomer units are covalently linked in the
dimer. Sedimentation velocity studies in the presence of low concentra-
tions of 2-mercaptoethanol showed that under these conditions the
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MOLECULAR SIZE
A N D
CONFORMATION OF IMMUNOGLOBULINS
339
dimer is converted to the monomeric species.
It is now established that two subclasses of human yA are present
in serum and certain external secretions (see below) and that the anti-
genic differences between yA, and yA, are associated with differences in
the
a
chains (D. Feinstein and Franklin, 1966; Kunkel and Prendergast,
1966; Vacrman and Heremans, 1966). This diffcrentiation was originally
detected on the basis of the antigenic deficiency of yA, when tested with
antisera prepared against y A , . In addition to its antigenic uniqueness,
yA,
has been shown to possess distinct chemical properties compared to
yA, . The a? and the light chains of yA, are not linked to each other
by
disulfide bridges and can be separated, in the form
of
stable disulfide-
linked dimers, in dissociating solvents without prior reduction (Grey
e t
u Z . 1968). The linking of the two light chains
in
the native molecule
by a disulfide bridge is an intriguing feature of the yA, structurc not
previously encountered in human immunoglobulins although a similar
situation occurs in certain murine yA-globulins
(
Abel and Grey, 1968).
The molecular size
of
the heavy and light chains of the yA,- and
yA,-globulins have been determined by Dorrington and Rockey ( 1970a,b)
by sedimentation equilibriuin in 6.0 A1 guanidine hydrochloride. The a
chain obtained from extensively reduced and alkylated yA by gel filtra-
tion on Sephadex G-200 in 8.0 M urea-0.05 hl propionic acid had a
molecular weight of 56,300
(S.D.
+1700). None of the preparations
examined showed any dependence of
A f
on initial protein concentration
or rotor speed. The light chain had a molecular weight of 22,700
(S.D.
t10 00) . The
0 1 ,
and light chains of yA,-globulin were examined by sedi-
mentation equilibrium in the form of disulfide-linked dimers obtained by
dissociating yA, in concentrated urea solution without prior reduction
and also as fully reduced and alkylated chains. Fully reduced a2 chain
had a molecular weight of 52,200 (S.D. t700) and the light chain,
22,800
(S.D.
k2400) . As anticipated the and light chain dimers had
molecular sizes approximately twice those of the monomeric chains, i.e.,
a2 dimer, 104,600 (S.D. 5 2 0 0 0 ) ; L dimer, 45,400
(S.D.
3~1500).These
data strongly suggest that the
N 2
chain is significantly smaller than the
N
chain by approximately 4000 gm./mole. Assuming this difference
is
due to changes in amino acid content rather than to variation in the mass
of carbohydrate, it represents a deletion of some thirty to thirty-five
residues. Work on the structure
of
yA, has not reached a stagc where one
can say how far this deletion is rcsponsible for the unique properties
of this subclass. It is not known whether the cysteine residue involved
in the hcxavy-light -S-S- bridge
in y A ,
is absent in
yA,
or forms an
intrachain -S-S- bridge. It
may
well be, however, that the deletion
occurs in the extended I"hinges") region of the a2 chain.
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340
KEITH
J ,
DORRINGTON
AND CHARLES
TANFORD
There seems to be an inconsistency in the molecular size
of
serum
yA cy chain determined by Dorrington and Rockey (1970a,b) and the
a-chain from rab bit colostral yA (C eb ra a nd Small, 1967). Although it
is possible tha t this differe nce is a real one, i t seems more likely to be
principally due to t he choice of 5 sed in the calculations. Dorrington an d
Rockey used a value of 0.718 calculated from amino acid and carbo-
hydrate analyses and adjusted for the effects of guanidine binding,
whereas Cebra and Small (1967) used a value of 0.732 based on direct
density measurements in
5.0
M guanidine hydrochloride. The latter value
seems somewhat high considering the values measured for intact yA
(0.685) and light chain (0.703) in 5 . 0 M guanidine hydrochloride by
the same authors. The interest in determining the real molecular size of
the a chain lies in whether
it
is larger than the y chain. If the value
of
56,300 (Dorrington and Rockey, 1970a,b) is adjusted for the bound
carbohydrate, we calculate 49,900 for the polypeptide portion of the
(Y chain, i.e., approaching the value for the y chain (48,600 ). Th e value
of Cebra and Small (1967) gives a polypeptide portion larger than the
y chain, which implies differences a t th e level of th e genom e, as sug -
gested for the
p
chain.
D.
OTHER
MMUNOGLOBULINS
Rowe and Fahey (1965a,b) demonstrated that a unique myeloma
protein was a representative of a new class of immunoglobulin (yD)
present in low, but variable, leveIs in normal human seium. Several
yD
myeloma proteins have been shown to have sedimentation coefficient
(s&,) between 6.1 S and 6.2 S (Row e
e t
al., 1969). Molecular weight
determinations, by the Archibald approach to equilibrium method,
yielded values near 183,000 (Rowe e t al., 1969) . A yD myeloma pro-
tein, studied in the laboratory of one of the authors (C.T.) by Eliza-
beth Rowe and R. Griffith was found to have a molecular weight of
abo ut 180,000 by sedimentation equilibrium. T he heavy chain
( 6
cha in) ,
by sedimentation equilibrium in 6
M
guanidine hydrochloride, had a
molecular weight
of
69,000. Another yD protein, studied by Dorrington
and Bennich (1970) gave molecular weights of 175,000 and 63,000 for th e
whole protein and the 6 chain, respectively.
An
unusual feature of these
proteins is the remarkably low1 sedimentation coefficient, in the light of
the relatively high molecular weight. The protein also seems to
be
un-
usually susceptible to the action of proteolytic enzymes (Griffith and
Gleich, 1970). A possible interpretation is that yD is less compactly
folded than other immunogIobulin molecules.
Bennich and Johansson
(1971 have reviewed the current knowl-
edge of YE, the most recently described class of imniunoglobulins.
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MOLECULAR SIZE AND
CONFORMATION
OF IMMUNOGLOBULINS
341
Therefore we will only briefly mention the most recent work on t he
molecular size of Y E a nd
c
chain.
Dorrington and Bennich (1970) found the molecular weight of
a
yE
myeloma protein to be 188,100 (S.D. +3000) from high-speed sedi-
mentation equilibrium studies. This is somewhat lower than the value of
196,000 obtained earlier by Bennich an d Johansson (19 67 ). T h e size
of t he c chain was found to be 72,500 (S.D. 22400) by sedimentation
equilibrium in 6 A 1 guanidine hydrochloride, of which 14%was accounted
for by carbohydrate. The polypeptide portion of the e chain (63,350) is,
therefore, significantly greater than for the heavy chains of other human
immunoglobulins.
I l l .
Conformation
o f
Immunoglobulins and Subunits
A.
OVERALL HAPEAND FLEXIBILITY
1.
yG-GLOBULIN
Two types of topographical model have been proposed for YG-glob-
ulin, incorporating the available information on the size and shape of the
molecular envelope, the localization of the antigen-binding sites, and
the contribution
of
the heavy and light chains to these sites. Edelman
and Gally (1964) considered the molecule to be a rigid rodlike struc-
ture with the antibody-combining sites at the extreme ends of the rod.
Noelken et al. (1 96 5) , however, favor a model in which most of the
polypeptide chains are incorporated into three compact globular regions,
corresponding to th e F a b an d F c fragments, l inked by a flexible exten ded
portion of the heavy chain. The models are compared in Fig. 1. T h e
properties of 7G upon which the evidence for which models are based
will be discussed in this section.
When the shape of a molecule is inferred from hydrodynamic data,
it must be realized th at such da ta can only provide evidence that th e
molecule is not globular.
It
cannot distinguish between an increase in
hydrodynamic radius due to a segment of randomly coiled polypeptide
chain and th at d ue to a nonspherical shap e with th e retention of rigidity.
If
the shape is nonspherical, it could be any regular or irregular shape
at all. Equations relating hydrodynamic properties to equivalent ellip-
soids of revolution have until recently bcen the only available equations
for quantitative interpretation, and ellipsoidal axial ratios, based on
these equations, have been frequently reported. These cannot be
ex-
pected to have m uch significance in terms of th e real molecular s ha pe .
From intrinsic viscosity, osmotic pressure, and sedimentation velocity
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342
KEITH J. DORRINGTON AND
CHARLES
TANFORD
Ligh t cha in
4
\
Fc fragment
FIG. 1. A
diagrammatic comparison of ( a ) the flexible model
of
yG
of
Noelken et al.
(
1p65) and ( b ) the rigid-rod model of Edelman and Gally (
1964).
In
both models the short, heavy lines represent the interchain disulfide bonds.
measurements, Oncley et
al.,
(1947) proposed that human
yG
was
approximately by a prolate ellipsoid with dimensions of 235 by 44A.,
assuming hydration of
0.2
gm. water per gram protein. A similar mo-
lecular length
(230A.
) was calculated from flow birefringence studies
on human yG in
60
to
70 %
glycerol solutions by Edsall and Foster
(1 94 8). This observation tends to supp ort the rigid rod m odel since
the Noelken-type structure should not show significant alignment along
the flow Iines
of
a viscous liquid. However, we do not know whether
the conformation of yG would be the same in concentrated glycerol solu-
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MOLECULAR SIZE AND CONFORMATION OF IMMUNOGLOBULINS
343
tion as in dilute salt and whether the presence of aggregates might
account for the birefringence properties. A series of low-angle X-ray
scattering studies by Kratky’s group (Kratky
et
at.,
1955, 1963;
Kratky,
1963) suggested a cylindrical model for human
y G,
with an elliptical
cross section and dimensions of 240
x
57
x
19 A. and total volume
The X-ray scattering method can distinguish between a molecule that
is completely randomly coiled and a molecule that is a completely rigid
ellipsoid, but whether it can distinguish the Noelken-type model from
a rigid rod is open to question. A recent pape r, representing th e combined
efforts of Kratky’s an d Edelman’s laboratories (P ilz et ul., 1970), at tempts
to answcr this question on the basis
of
new measurements using
a
homo-
geneous myeloma
y G.
Theoretical scattering curves for eight different
models are compared to the experimental scattering curve, and it is
concluded that the best agreement is obtained with a rigid T-shaped
model, in which there is no central region of relatively low density of
scattering material, as is shown in the model of Noelken et
al.
It is not
easy to decide how seriously this conclusion should be taken, especially
as the molecular weight determined from the same data is in error by
10%.The myeloma protein used for this study is that for which the
complete amino acid sequence has been determined (Edelman
et
d. ,
1969), and the true molecular weight is 148,000. The experimental value
was 162,000.
A comparison of the hydrodynamic properties of intact
yG
and the
Fab
and Fc fragments produced during limited proteolysis with papain,
provided some of th e evidcnce for th e “flexible” mo del of Noelken
et
al.
(1965) .
The fractional coefficient ratios (f/flll,,,) were calculated for
yG
and t he fragments to give a measure of deviation from a compact g lobular
shape. The ratio, f/fIrl,,,, is a measure of the combined effects of hydra-
tion and shap e on hydrodynamic properties. Fo r typical globular proteins
flf,,,,,,ies in the range of 1.10 to 1.25, indicating that both hydration and
deviation from a spherical shape are small. Fragments Fab and
FC
behave as typical globular proteins f/flllI,,
= 1.21-1-24),
whereas intact
7G does not
f / f i l l ,
/ = 1.47) . Therefore either yG is more asymmetrical
or possesses
a
large amount of hydration. Hydration in this context in-
cludes hydrodynamically trapped solvent, so that a molecule with regions
of flexibly coiled polypeptide chain would appear to have anomalously
large amounts of hydration when examincd by hydrodynamic methods.
The same conclusion is reached from intrinsic viscosity measurements.
Intac t human and rabbi t
7G
has
[ T I ]
= 6.0 cc./gm. (Jirgensons, 1963;
Noelken
et
ul., 1965), whereas the values for the papain fragments are
near 4.0 cc. /gm . These findings arc’ coinpatiblc, with
a
stru ctu rc consisting
2.0 x 105
i i . 3 .
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344
KEITH J . DORRINGTON AND CHARLES TANFORD
of compact Fab an d Fc regions linked by a flexible, extended portion con-
sisting of a part of each heavy chain but does not exclude a rigid linear
arrangement of the fragments ( i .e., Fab-Fc-Fab)
.
The most convincing evidence in favor of the flexible model is the
observation that the heavy chains of yG can be cleaved in a relatively
limited region by a variety of proteolytic enzymes and cyanogen bromide.
T he region of th e heavy chain in w hich th e flexibility proposed by Noel-
ken et
aZ.
must occur (so-called “hinge” region) has been delineated by
the amino acid sequence studies of Smyth and Utsumi
(1967)
and of
Givol and D e Lorenzo (19 68 ). Th ese workers h ave defined th e cleavage
points for papain, pepsin, typsin, and cyanogen bromide (Fig. 2) . T he
region of the chain containing these cleavage points must
be
much more
freely accessible to cyanogen bromide and to the active sites of the
proteolytic enzymes than similar susceptible bonds elsewhere in the yG
molecule. It is interesting that the region involved contains three con-
secutive prolyl residues, immediately carboxy terminal to the inter-heavy-
chain disulfide bond, on each heavy chain. Such a prolyl tripeptide is
unique among known sequences and may accout, in part, for the
extended n atu re of this region of the heavy chain. Givol an d D e Lorenzo
(1968) limit the flexible region of the heavy chain, accessible to pro-
teolytic enzymes, to between
25
and 30 residues on each chain.
A flexible model for yG-globulin seemed to provide an explanation
for the low values of the rotational relaxation time obtained from
fluorescence depolarization studies. Dimethylaminonaphthalene sulfonyl
chloride ( D N S ) conjugates to yG have been studied by a number of
workers (Churchich, 1961; R. F. Steiner and Edelhoch, 1962; Chowd-
hury and Johnson, 1963;
M.
H. Winkler,
1965)
an d have provided values
of less than 100 nsec. for th e relaxation time . O n th e basis of t he
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MOLECULAR SIZE
AND
CONFORMATION O F IMMUNOGLOBULINS
345
molecular dimensions obtained from other measwenients, when in-
terpreted in terms of a rigid ellipsoid, a value near 220 nsec. would have
been an ticipated. Ind eed , measu remen ts of th e rotational relaxation time
fo r horse yG by dielectric dispersion (Oncley, 1943) and human yG by
electric birefringence relaxation ( Krause a nd O’Konski, 1965 ) have given
values of 220 and 200 nsec., respectively. Electric birefringence studies on
bovine yG gave rotational relaxation times of 215 nsec. (In gra m and
Jerr ard , 1963 .) T he discrepancies in the relaxation times p rovided by
fluorescence depolarization measurements and other methods was in-
terpreted to mean tha t diffe ren t regions of th e yG molecule could rotate
independently; a situation which could be envisaged in the Noelken
et
al.
model.
More recently this interpretation of the fluorescence depolarization
data has been challenged
(Wel tman and Edelman, 1967; Wahl and
W eber, 19 67 ). These workers suggest th at thermally activated rotations
of the covalently bound DNS groups can occur independently of the
rotation of the region of the molecule to which they are attached thus
leading to low values for the rotational relaxation time of the protein.
The use of fluorescence depolarization to determine molecular relaxation
times depends on the requirement that the fluorescent label should be
immobilized by strong interactions with the protein molecule. Weltman
and Edelman (1967) and Wahl and IVeber (1967) showed that the
slopes of the fluorescence depolarization curves obtained isothermally
at varying viscosities (sucrose isotherms) are smaller than the slopes
obtained when the temperature is changed to alter the viscosity of the
solvent witho ut sucrose. Values of t h e rotation al relaxation time c alcula ted
from sucrose isotherms a t different temp eratures ranged from 19 1 to 244
nsec., wherea s he atin g curves gave values below 100 nsec. in agreem ent
with earlier studies. These results seemed to indicate that under experi-
mental conditions where free rotation of the dye molecules is minimized,
values of the rotational relaxation time c i n b e obtained from fluorescence
studies which are apparently consistent with values obtained by other
methods and previously calculated molecular dimensions. This does not
appear to
be
the whole story according to a recent paper by Zagyansky
et al. (19 69 ). Th e lat ter have shown that dimethylaminonaphthalene
sulfonyl chloride ( D N S ) conjugates of human and rabbit yG have dif-
ferent fluorescence properties from DNS conjugates of serum albumin
and ovalbumin. Of particular relevance was the finding that the lifetime
of the excited state for DNS -yG was shortcr (7. 3 nsec.
)
than for DNS
albumin (12.1 nse c.). Since the value
of
thv lifetime of the excited state
(.) is required in thc depolarization calculations, exact knowledge of it
is of great importance. Zagyansky et 01. (1 96 9) calculated thc rotational
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346
KEITH J .
DORRINGTON AND
CHARLES TANFORD
relaxation times of the DNS yG to be near 60 nsec. using sucrose iso-
therms and T = 7.3 nsec. Recalculation of t h e da ta of W eltman an d
Edelman
(1967)
and Wahl and Weber
(1967)
gives values of
100
to
130 nsec. using the lower lifetime value, The even lower value of
60
nsec. obtained by Zagyansky
e t
al. may hav e been due, in part , to their
removal of aggregates from the DNS yG preparations before the fluores-
cence measurements. These authors also provide some experimental
evidence that th e shorter lifetime of th e excited state in D NS yG may
be explained either by the substitution of DNS onto different amino
acid side chains, the alternative conjugates having different spectral
properties, or by differences in the hydrophobic nature of the environ-
ment of the DNS groups between DNS
yG
and DNS BSA. DNS-aspar-
tate transaminase seems to have similar fluorescence lifetime properties
to DNS yG (Polyanovksy et al., 1970).
It seems from the above discussion that, even allowing for free rota-
tion of the dye molecules, the rotational relaxation time for DNS yG is
lower than expected for a rigid molecule. Thus it would seem that the
units
of
the yC molecule with some rotational freedom are smaller than
the whole molecule, and it is tempting to suggest that they correspond
to the Fab and Fc fragments.
Two recent papers have reported fluorescence polarization measure-
ments by direct measurement of the relaxation process (time span in the
nanosecond range) following excitation by very short light pulses. This
technique is free from some of the ambiguities that apply to previous
results obtained by steady state measurements under constant illumina-
tion. Wahl (1969) employed DNS yG, with the DNS coupled covalently
to the protein. His results were analyzed in terms of two relaxation
processes, with relaxation times of
370
and
23
nsec. The first, which
accounts for 65%of the overall relaxation process, is ascribed to rotation
of the whole molecule and the second to internal Brownian movements
of a globular region of the molecule. ( T h e relaxation time, howeve r, is
m uc h too sho rt to repre sent a globular region of t he size of
the
Fa b region
of the molecule. )
The second paper using the nanosecond fluorescence technique is
by
Yguerabide
et
al.
(1970). They coupled the DNS chromophore to yG
noncovalently by using yG that was a n antib ody directed against i t. They
also observed two relaxation times, one of
500
nsec., which is of t he same
ord er of mag nitude as Wahl’s longer relaxation time. T he shorter relaxa-
tion time, however, was found to be 100 nsec., i t . , four times as large as
WahPs. W hen th e same measurements were carried ou t with the complex
betw-een the DNS hap ten an d th e Fa b fragment of th e antibody, only a
single relaxation time of 100 nsec. was observed. The 100 nsec. relaxation
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MOLECULAR SIZE AND CONFORMATION
O F
IMMUNOGLOBULINS 347
time is consistent with expectation for
a
rigid ellipsoid, molecular weight
50,000
plus hydration of 0.32 cc ./gm . with an axial ratio of a bo ut 2. These
are t he pro bab le dimensions
of
the Fab fragment, which thus appears to
rotate as a n essentially rigid u nit. T he rcsults as a whole, including addi-
tional da ta for
F(
ab '),, are completely consistent with th e flexible model,
but inconsistent with a rigid ellipsoid model for the native yG molecule.
Electron microscopy has been widely used to stu dy the size and sh ape
of yG-globulins despite the uncertainties inherent in the extrapolation
from the dehydrated state to proteins in solution. Observations have
been made either directly on immunoglobulin preparations or on anti-
bodies attached to large particles which can be clearly seen on negative
staining.
A
review of the subject has appeared recently (Green,
1970)
and we will concern ourselves only with certain aspects of the data.
Recent electron-microscopic studies have provided evidence that 7G
is m ad e up of thr ee linked globular regions.
A .
Feinstein an d Rowe (1965)
first suggested, from negative contrast studies on ferritin-antiferritin
complexes, that when cross-linking of antigen occurs the antibody mole-
cule "clicks open" to varying degrees about a hinge point located at one
end. A significant technical advance was made by Valentine and Green
(1967)
who studied complexes of purified high-affinity antibody to 2,4-
dinitrophenyl
( D N P )
with
a
bifunctional
D N P
hapten
(DNP-NH.
(
C H , ) s . N H . D N P ) .
On mixing equivalent amounts of
the
antibody and
hapten, symmetrical cyclic polymers are formed. The number of anti-
body molecules in the polymer depends on the angle between the com-
bining sites (e .g ., an ang le of 60" gives trimers of th re e divalent molecules
and 90 gives tetramers).
The
advantages of this syinmetrical arrange-
ment are many: the shape
of
the polymer indicates the arrangement of
the binding sites and the common features of each unit can be distin-
guished from the random distortions caused by the negative staining.
In addition, the units are coplanar and are viewed in the same orienta-
tion, relative to the electron beam. From such studies, Valentine and
Green (1967) an d Valentine (1967) conclude tha t the antibody molecule
is flexible since a wide ra ng e of different shapes ar e formed. Th e overall
morphology of the YG molecule is Y-shaped m ad e up of three rigid rods
representing th e F ab
and
Fc fragments (Fig. 3 ) . Th e angle be tween the
two Fab fragments is apparently variable between nearly
0"
and
180".
On the electron micrographs the Fc fragment is seen as a conspicuous
projection a t th e corners of the various figures (Fig. 3) . Incubation with
pepsin leaves the geometrical figures intact but removes the projections,
thus confirming that they are
Fc
fragments. The antigen-binding sites
lie at the extreme ends
of
the Fab fragments since the various shapes
show no distortion where the molecules are linked by the hapten. The
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348
K E I T H
J .
D O R R I N G T O N A N D CHARLES T A N F O R D
FIG.
3. Electron micrograph
of
complexes of rabbit yG antibody, directed
against the 2,4-dinitrophenyl
(DNP)
group, with the bifunctional reagent bis-N-
DNP
octamethylenediamine (Valentine and Green,
1 9 6 7 ) .
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MOLECULAR SIZE AND CONFORMATION OF IMMUNOGLOBULINS 349
maxiinum length
of
the Fab fragments was 70 A, thus making the maxi-
mum span of the molecule approximately 140 A.
Pilz
et
al.
(1970) point out that the actual dimensions of the Valen-
tine-Green model, which lead to a calculated radium of gyration of 46 A.,
are inconipatible with the radius of gyration (76
A . )
determined in
solution from X-ray scattering measurements. They suggest that the
discrepancy arises from dehydration and partial “collapse” of the original
antibody-hapten complex on th e surface of the grid used to support the
sample for electron microscopy. Such
a
constriction of th e molecule would
seem to be most likely to occur if the greater extension of the molecule
in solution arises from the presence of a region of the polypeptide chain
that is loosely coiled. It is more difficult to visualize at what portion of
the molecule the constriction occurs if one accepts the model for the
structure in solution preferred by Pilz
et
nl.
Further evidence for a Y-shaped model for
YG
has recently co me from
transient electric birefringence studies on a rabbit high-affinity anti-DNP
by Cathou a nd O’Konski (1 97 0) . Th e birefringence of an an tibody
solution was measured in the presence of zero, one, and two moles of a
tribasic ha pte n, a-dinitrophenyl glutamy l aspartate, pe r mole of antibody
and found to be the same in all three cases. The results were not com-
patible with a rigid-rod model. On the basis
of
comparisons
of
calculated
and experimentally determined specific Kerr coefficients, the authors
favor
a
Y-shaped model where th e angle between the F ab fragments lies
between
130
an d 180 degrees.
It has been recently established that there are additional positions of
preferred cleavage
by
proteolytic enzymes at positions halfway between
the hinge region
of
the yG molecule an d th e chain termini. Although the
rate of cleavage at these points is much slower than at the hinge region,
it is measurably fast. Thus fragm ents of lig ht chain (m ole cu lar weight
11,000-12,000
)
occur in t he urine of persons having Bence- Jones pro teins,
and presumably correspond to the variable and constant halves of the
chain (Deutsch, 196313; Solomon et nl., 1966; Williams et
al.,
1966; Cioli
and Baglioni, 1966; Van Eyk and Myszkowska, 1967; Tan and Epstein,
1967). Solomon and McLaughlin ( 1969) have demonstrated splitting
of
Bence-Jones proteins by endogenous urinary endopeptidases at acid pH,
and also showed that light chains could be split into V and C regions by
papain, pepsin, trypsin, and subtilisin. Bjork (1970) has shown that
incubation of isolated rabbit heavy chains with papain results in the
production of small amounts of a fragment with a molecular weight near
12,000. In ad dition fragmen ts corresponding to th e carboxy terminal half
of the Fc fragment have been isolated from pepsin and papain digests of
yG (T urn er an d Bennich, 196 8). Karlsson
et
nl. ( 1969) have shown that
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350
KEITH J . DORRINGTON AND CHARLES TANFOHD
the halves of light chains either occurring naturally in urine or produced
by proteolysis
in
vitro have compact globular structures. Such fragments
hav e molecular we ights of 10,000 to 11,000, from sedim entation equi -
librium studies, and sedimentation rates near 1.6 S, which, when com-
bined, yield a frictional ratio near 1.1. A Stokes radius of 1 6 A was
calculated from their behavior on gel filtration.
This information suggests that the F ab an d F c fragments a re not th e
basic compact domains of the
yG
molecule b ut tha t the compact domains
correspond t o th e partly homologous segm ents of about 110 residues th at
constitute th e bu ilding blocks of the am ino acid sequence. Each segment
is folded into a tight globular structure, but there are relatively un-
structured regions between segments, smaller in extent a nd less accessible
at the junction points within th e F ab and F c fragments than a t the hinge
region in the center of the molecule. In our opinion, an extension of the
flexible model, proposed by Noelken et al. (19 65) , to incorporate th e
probable existence of smaller compact domains and additional “loose”
connecting regions, would account in the most satisfactory way for all
+
Fc
FIG. 4.
The “compact domain” model for yG. Each circle represents a com-
pact, globular region of polypeptide chain
of
about 11,000 molecular weight and
containing
a
single intrachain disulfide bond. The heavy arrow indicates the region
(h inge )
of
the heavy chain most susceptible to enzymatic and chemical cleavage.
The interrupted arrows indicate the sites on both the heavy and light chains which
are sensitive to cleavage but to a much lesser extent than the hinge region. (Adapted
from Edelman and Gall, 1969.)
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MOLECULAR SIZE
AND
CONFORMATIOIV
OF
IMMUNOCLOBULINS 351
the experiinental results cited in this scction. A representation of the
molecule on this basis is shown in Fig.
4,
taken from a recent review by
Edelman and Gall (1969). In view of the fact that these authors are also
co-authors of the paper by Pilz et
al.
(1970), cited earlier,
it
is likely that
their opinions as to the exact nature of the connecting regions differ some-
what from those of the authors of this review. However, it is clear that
the major difference in interpretation inherent in the two models shown
in Fig.
1
no longer exists.
2.
yAi-Globulin
Hydrodynamic studies on Waldenstrom macroglobulins clearly indi-
cate that
y M ,
like yG, shows gross deviations from a compact globular
structure. Calculations of the frictional coefficient ratio
( f/f,,,l,l )
from
sedimentation and diffusion constants for intact
yM
and 7 S
yM,
gave
values of 1.92 and 1.69, respectively ( Miller and Metzger, 1965a). VaIues
of the intrinsic viscosity for yM have varied from 6.0 (Kovacs and Daune,
1961) to 20.0 cc./gm. (Jirgensons et al., 1960). In a careful study Jahnke
e t
al. (1958)
obtained
a
range from 10.6 to 15.3 cc./gm. for four dif-
ferent yM preparations. Miller and Metzger (1965a) obtained 16.2
cc./gm. for their yM which dropped to 8.0 cc./gm. upon mild reduction
to yM,. These high valurs for the intrinsic viscosity are consistent with
the elevated
f / f l l l l l l
ratios. The wide range of values for the intrinsic
viscosity may reflect various degrees
of
polymerization of the 19 S species
in the yM preparations used. Aggregation may also account for the
variations in
&, 2o
and
D & ,
values found for different YM-globulins
(Suzuki and Deutsch, 1967).
The high values of the frictional ratios and the intrinsic viscosities of
yM can be accounted for by either
a
rigid or a flexible model for yM as
was the case for yG. Metzger
et
al.
(1966) used fluorescence depolariza-
tion studies with DNS conjugates of yM in an attempt to distinguish
between these two alternatives. A mean rotational relaxation time of
1700 nsec. was calculated for a hypothetical prolate ellipsoid with an
axial ratio of 18 (i.c., consistent with the hydrodynamic data). However
the experimentally determined relaxation time for the
DNS y M
was only
80
nsec., nearly an order of magnitude lower than the theoretical mini-
mum value for a sphere of equivalent mass (730 nsec.). The short relaxa-
tion time of yM was not simply due to independent rotation of the yM,
units, since these units had a relaxation time of 69 nsec. which is less than
that expected of a sphere of equivalent mass. This suggests that
yM,
also
has considerable internal flexibility. The relaxation time of the tryptic
fragment of yM, Fab
p,
was greater than expected for an equivalent
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352
KEITH J. DORRINGTON AND CHARLES TANFORD
sphere, suggesting that the internal flexibility of this unit is negligible.
Th e relaxation times of F ab /L and intact yM were closely similar suggest-
ing that the regions corresponding to Fab
p
represent the largest rota-
tional subunit of yM.
As
in some of th e earlier studies on th e fluorescence
depolarization of
yG
(Section 111,AJ ), Metzger et
al.
make the assump-
tion that the dye molecule interacts sufficiently strongly with the region
of
th e protein to which it is attac he d so tha t it truly reflects the rotational
properties of th at region. T he objections raised concerning the in terp reta -
tion of the depolarization studies of yG because of thermally activated
rotations of the dye molecule, independently of the protein, may be
relevant to the studies on yM. Reinvestigation of the relaxation proper-
ties of
yM
under isothermal conditions at different solvent viscosities,
together with measurements
of
the lifetime of the excited state, seem to
be indicated fo r yM.
Some interesting observations regarding the ultrastructure of yM
have appeared recently from electron micrographs (Svehag e t al., 1967;
Chesebro et al., 1968; A. Feinstein an d M unn, 19 69) . Examination of
negative contrast preparations of yM from a variety of species revealed a
high concentration of stellate structures (Fig. 5 ) . These structures ap pear
to be composed of five “legs” connected to a central ring. The legs are of
variable length, apparently bent in different configurations, thus giving
particles of different diameters. The average diameter or span is 300A.
and each leg has maximum dimensions of 100 x 25 A. The central ring
has a n outer diameter
of
100
A.
an d a hole of
40
A. diameter. A. Feinstein
and Munn (1969) provide evidence that the legs are divided into two
FIG.5 . Electron micrographs of individual yM molecules visualized by negative
staining. On the left is the more commonly seen figure showing the five “legs” joined
to
the central ring; on the right, a figure with each leg divided into two. (Reproduced
by
kind permission of Drs. Feinstein and Munn.)
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MOLECULAR SIZE AND CONFORMATION OF IMMUNOGLOBULINS 353
along part of their length (55-70
A ) .
Thcse tw o regions of e ach leg ar e
thought to represent the Fab /L fragments, and where they join represents
a
flexible hinge region which accounts for the variable disposition in
space of the legs seen in the micrographs. Further evidence for the
flexibility of this region has been provided by these workers: electron
micrographs of antibody-antigen complexes (
SaZmoneZZu
flagellum-yM
antiflagellum) show yM molecules with all their legs bent around and
attach ed to th e flagellum.
The electron-microscopic evidence together with the chemical infor-
mation available on yM can be used to construct a schematic model for
this m olecule (F ig. 6 ) . Th e model is based on the following chemical
evidence:
a. Molecular weight and other evidence indicates that yM is made up
of five units
(
Y M ,) of m olecular size 180,000 (Ta b le I ) .
b. Enzymatic cleavage with papain produces an Fab
p
fragment,
molecular weight
48,000,
composed of one light chain and part of the
amino-terminal region of the p chain (Mihaesco and Seligmann, 1968a;
Dorrington an d Mihaesco, 1970 ). Each Fa b
p fragment has a single
antigen-binding site. Papain also produces an Fc
p
fragment, molecular
size 320,000, which yields, on reduction in 6 M guanidine hydrochloride,
fragments of mass near 32,000 (Dorrington and Mihaesco, 1970) indicat-
ing that it is mad e up of the carboxy-terminal regions of the ten
p
chains.
It seems likely that the central ring of yM seen in the electron micro-
graphs represents this polymerized Fc
p
fragment.
c. Pepsin and trypsin produce a fragment of molecular size near
120,000 (
F
( a b” )
p )
with two antigen-binding sites
(
Mihaesco and Selig-
mann, 1968b; Miller and Metzger, 1966; Metzger, 1967). This fragment
is composed of two light chains and a region (Fd”) of each of the heavy
chains of molecular size near 36,000. T he F( ab ’’) 2p is fur the r digested b y
pepsin to give two Fab”
p
fragments of similar molecular size and chain
composition to papain Fab
p
(Mihaesco an d Seligmann, 1968b; Miller a nd
Metzger, 19 66 ). Chymotrypsin
C
digestion yields a larger fragment, of
mass 135,000 (Chen
et
al., 1969) .
d. The mass of yM can be accounted for by the polymeric Fc
p
and
five pepsin
( F (
b”)?) ragments. Dorrington and Mihaesco (1970) have
delineated three antigenically distinct regions of the
p
chain; Fd
p
( o r
Fd’
p )
with a mass near 24,000, F c
p
with a mass of approximately 32,000,
and a region not included in these two fragments which is very sensitive
to proteolytic attack. This latter region is reminiscent of the extended,
flexible hing e reg ion of yG. In add ition to its sensitivity to proteolysis this
region shows only a low level of conformational antigenic determinants
(Mihaesco an d Seligmann, 196Yb). T hc pu tative hinge region of yM is
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354
KEITH
J . DORRINGTON AND CHARLES TANFORD
Papain
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MOLECULAR SIZE AND CONFORMATION OF IMMUNOGLOBULINS 355
approximately 80 to 90 residues, significantly longer than the correspond-
ing region of the y chain. The Fd
p
regions are probably com pact an d
globuIar since they are fairly resistant to proteolytic attack. The
Fc
p
is
probably not as tightly folded since proteolysis can occur fairly readily
in this region of the p chain, Fc
p
being ob tained in low yield. A fragmen t
corresponding to Fc p is not obtained from yM, suggesting that much of
the resistance to papain is a function of the close apposition of the sub-
units in intact yM.
e. T he yM, units are joined by a single disulfide bond, which is shown
as th e m ost carboxy-terminal half-cystine residue
of
t he
p
chain in Fig. 6.
More recent evidence indicates that the adjacent
(
more amino-terminal )
half-cystine
is
involved in the inter-yM, disulfide bond (B ea le an d Fein-
stein, 1969,1970).T he cyclic arrangem ent of th e yM, subunits in intact y M
is clearly shown in th e electron micrographs b u t was suggested by earlier
studies on the disulfide bonds
of
yM (M iller an d M etzger, 196513). I t is
also difficult to understand why simple linear polymers of yM, should
be restricted to
f ive
units unless a cyclic structure is formed. Presumably
a pentameric rin g structure is the m ost thermodynamically stable. D uring
the biosynthesis of
Y M,
the polymerization of YM, seems to occur just
prior to or simultaneously with secretion from the cell since only
yM,
can be detected w ithin the cell (Parkhouse an d Askonas, 196 9). Th ere
is no evidence for the secretion of intermediate polymers
of
yM,.
Considerable attention has been focused on the intriguing question
of
the valency of yM antibodies. A number of studies indicate that yM
antibodies possess five bind ing sites-one associated with each
y
M,
subunit (Onoue et
al.,
1965; Lindqvist and Bauer, 1966; Metzger, 1967;
Stone and Metzger, 1968; Coligan a nd Bauer, 1 96 9). How ever, i t has
been demonstrated for one such antibody, a Waldenstroni macroglobulin
with antihuman YG activity, that each tryptic F ab p fragment can bind
antigen with equal affinity (Metzger, 1967; Stone and Metzger,
1968).
Clearly, then, this antibody had ten potential binding sites, only half
of
which were available in the intact yM. Ry contrast, other yM antibodies
have been described where ten binding sites were detectable in the 19 S
molecule (Merler ef nl., 1968; Onoue et nl., 1968). The antihapten yM
studied by O noue et al. (1968) ha d tw o groups of binding sites, five with
affinities 100-fold greater th an the other five. Each yM. ap pe are d to
possess one high and one low affinity site. In the antibody studied by
A model for
yM
based on
the
electron-microscopic evidence (upper)
and chemical evidence (low er ). The upper part
of
the figure
is
a clay scale model
constructed from dimensions obtained from the electron micrographs. The lower
part of the figure is based on a variety of chemical observations (for discussion see
text).
FIG. 6.
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356
KEITH
J .
DORRINGTON AND CHARLES TANFORD
Merler et
al.
(1968) , all ten sites were of equal affinity. The reason for
the unavailability of one of the two paired sites on yM, is not known at
this time.
It
is possible that th e phenomenon might
be
explained b y steric
hindrance; antigen bound to one site preventing access to the neighbor-
ing site. This may well account for th e situation in antibodies to m acromo-
lecular antigens (e.g., yG) but is not
so
convincing for low molecular
weight haptens. Alternatively, the quarternary folding of yM might lead
to a situation where only half the binding sites are in a suitable con-
figuration to allow significant interaction with the antigen.
It is interesting to note in this connection that the 7 S and 19 S yM of
elasmobranchs differ in valency. The naturally occurring 7 S-yM anti-
body is apparently bivalent since
it
can agglutinate antigen-coated
erythrocytes and the activity is resistant to further mild reduction. How-
ever the 7 S yM, obtained following mild reduction of the 19
S
species
shows no activity in the same test system (Clem and Small,
1967).
T he
structural and biosynthetic bases for this difference in valency between
the two 7 S species remain to be determined,
3
INTERNALOLDING
A t the present time X-ray crystallography provides the ultimate in
protein conformational analysis, and its usefulness has been admirably
demonstrated over the past several years ( Davies, 1967; Stryer, 1968).
It is possible with this technique, under optimal conditions, to describe
with a high degree of accuracy the secondary and tertiary structure of a
protein. Until very recently the possibility of performing such an analysis
on immunoglobulins seemed very remote; the heterogeneity of these
proteins seemed to preclude the preparation of stable, suitably sized
crystals for analysis. The crystallizability of rabbit yG Fc f ragment has
resulted in some preliminary observations of its crystal properties ( PoIjak
et al.,
1967;
Goldstein et al.,
1968).
However, while F c has a number of
interesting biological properties, studies on this fragment are unlikely to
answer the vital questions regarding the relationship between protein
structure and antibody activity.
T h e recent discoveries of crystalline 7-globulins, a hum an yG myeloma
protein (Terry et al., 1968), a myeloma yG Fab fragment (Rossi and
Nisonoff, 1968), an d a rab bit antiazobenzoate antibody (Nisonoff et al.,
1967) suggest that X-ray examination of intact immunoglobulins may be
possible. I n fa ct
a
preliminary report has appeared on a hum an myeloma
VG cryoglobulin (T er ry et
al.,
1968). Unit cell dimensions have been
measured an d evidence obtained for half a m olecule per unit cell, indicat-
ing that yG has a twofold axis of symmetry. The presence of a diad axis
is consistent with the chemical data showing th at th e molecule is m ade u p
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MOLECULAR
SIZE AND CONFORMATION OF IMMUNOGLOBULINS
357
I
I
____---------
-
-
I
I
I
1
I
I
I
-
1 I I I I I I
I
of two identical pairs of chains linked by disulfide bonds. Also the Fc
fragm ent has been shown to have twofold symmetry (Goldstein et al.,
1968).
T he crystalline
Fc
can be enclosed in a parallelepiped
50
x
40
x
70 A.
When these dimensions are corrected for the water content of the
crystal, they are consistent with those obtained from the electron
micrographs.
In the absence of detailed X-ray analysis, much of the information on
the conformation of immunoglobulins i n solution, although speculative,
has come from opticaI rotatory dispersion (ORD) and, more recently,
circular dichroism (CD) studies. Discussion of the theory and measure-
ment of these two related phenomena will not be attempted here since
excellent reviews have ap pear ed elsewhere (U ni es and D oty,
1962;
Yang,
1967;
Beychok,
1967, 19 68 ).
Much of the ORD data o n immunoglobulins has been performed on
yG-globulin. The spectra of yG from a variety of species are essentially
-
:
FIG.7 .
The optical rotatory dispersion spectra of a human yC myeloma protein
(
H u - y C )
and a sample of pooled rahbit
yG
R b - y C ) between 300 and 200 mp
Inset shows the
260-220-mp.
region in more detail. These two spectra illustrate the
kind
of
variability foimd
i n
the opticlil rotation
of
difierent preparations of
yC.
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358
KEITH J . DORRINGTON AND CHARLES TANFORD
similar (L. A. Steiner and Lowey, 1966; Dorrington
et
al., 1967; Cathou
and Haber, 1967; Ross an d Jirgensons, 1968; Rockey
et al.,
197 0). Basi-
cally the ORD of yA and yM show similar features to the spectra
of
yG
( Dorrington and Rockey, 1968; Dorrington a nd T anford, 1 968 ). Th e
spectra of these immunoglobulin classes are characterized by ( a ) a low
level
of
rotation throughout the wavelength range,
( b )
Cotton effect
minima near 230, 225, and 198
mp.,
( c ) a single maximum between 204
an d 210 mp. with a crossover ( ze ro rotatio n) nea r 220 mp., an d ( d ) only
in
yG
a Cotton effect of low rotatory streng th at 240 mp. (Fi gs. 7-9).
The absolute levels of rotation
at
the maximum and minima show con-
siderable variation betw een different proteins w ithin an y imm unoglobulin
class.
More recently, attention has been focused on the circular dichroic
I
Wavelength ( m p )
FIG.8. The optical rotatory dispersion spectrum of human yhl-globulin (solid
line) compared with hmnan y
G ( d a s h e d
line) between 200 and 400 ma. Inset
shows the
2207300-nip.
region in
more
detail. ( From Dorrington
and
Tanford,
1968.
)
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MOLECULAR SIZE
Ah?)
CONFORMATION OF
IMMUNOGLOBULINS
359
200
240 280
320 360
4
Wavelength
0
FIG.
9. The optical rotatory dispersion spectrum
of
a human
yA
myeloma
pro-
tein (solid line) compared with human yG (dashed line) between
200
and
400
mF.
Inset shows the 220300-mg. region in greater detail. (From Dorrington and Rockey,
1968.)
properties of yG (Ca thou e t al., 1968; Iked a et al., 1968; Ross an d Jirgen -
sons, 196 8). Although circular dichroism is closely related to O R D, th e
optically active transitions are seen as discrete bands usually set against
a background of zero optical activity. The component bands are more
easily resolved th an t he individua l Cotton effects from th e O R D spectrum.
Th e most extensive study of th e C D of yG has been performed by Cathou
e t at . (1968) using rabbit high-affinity anti-DNP. The CD spectrum of
yG
(Fig. 10) exhibits negative bands at 192, 217, and 240 nip.; positive
bands are seen at 202 and 232 mk. and in the 260300-nip. region. The
transitions of low rotatory strength above 260 mp. can be partially
resolved at 275 to 280 and
290
mp.
T h e O R D
of yG
and y M can be reproduced by
an
approp riate com-
bination of the spectra of their respective Fab and Fc fragments indicat-
ing that little change in conformation occurs during proteolysis ( L . A.
Steiner an d Lowey, 1966; Dorrington an d Tanford, 1 96 8). Th e O R D
spectra
of
Fab and Fc f rom yG are shown in Fig.
11.
The 225- and
24O-nip. Cotton effect minima of
y C
are due to Fab, whereas the 230-
232-iqL. minimum can be at tr ibuted to the
Fc
region. These minima of
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360
KEITH J.
D O R R I N G T O N A N D C H A R L E S T A N F O R D
FIG. 10. The circular dichroism spectrum of a human y C myeloma protein,
between 200 and 300 ma., in 0.1 M NaCI-O.01 M phosphate buffer,
pH
7.2. Note
the difference in scale between the right- and left-hand ordinates.
Fab and Fc between 225 and 235 mp. probably represent optical activity
associated with the
n+ T
electronic transition of the peptide chromo-
phore, by analogy with synthetic polypeptide spectra. The reason for the
marked blue shift of this transition in Fab is not understood. The addi-
tivity
of
the spectral properties of Fab and
Fc
fragments in intact
yG
has been confirmed by CD (Cathou et
al.,
1968). It is worthy of note
that although the ORD spectra of intact
y M
and yA do not exhibit the
small Cotton effect at 240
mp.
(Dorrington and Tanford, 1968; Dorring-
ton and Rockey, 1968) the Fab and F(lab’)2 fragments derived from
these proteins clearly show this feature. In the case of
y M
the failure to
detect the 240-mp. minimum was due to the larger contribution of the
Fc region to the total rotation of the whole molecule. The 240-mp. Cotton
effect seems to be a common, characteristic feature of all the immuno-
globulin classes studied so far (all except
7D)
with the exception of
equine yT (Rockey
et al.,
1970). The optical transition responsible
for the 240-mp. Cotton effect is not known at this time but the possible
chromophores which may be involved have been discussed (Cathou
et
al., 1968; Rockey
e t al.,
1970).
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MOLECULAR SIZE
AND CONFORMATION OF IMMUNOGLOBULINS 361
FIG.
11.
The optical rotatory dispersion spectra
of
a
human
y C
myeloma pro-
tein and the Fab and Fc fragments isolated from a papain digest of the same protein.
Th e contributions of th e heavy a nd light chains to the O R D spectrum
of yG will be discussed in Section II1,C. The results suggest that the
225-mp. minimum of the whole molecule and its Fab fragment arises
principally from t h e light chain, whereas th e 240-mp. Cotton effect comes
from the heavy chain. The 232-mp. minimum is seen in heavy-chain
preparations, which is consistent with its presence in the
Fc
fragment.
Although the OR D and C D properties of immunoglobulins ar e
reasonably well established over the experimentally accessible wave-
length range, interpretation of these features in terms of specific con-
formations is impossible at this time. It has become fashionable to try
to account for the ORD an d C D properties of globular proteins in terms
of fractional contents of
a
helix,
p
structure, and random coil on the basis
of th e OR D and C D of synthetic polypeptides known to b e in these
conformations (Greenfield et
ul.,
1967; Magar, 1968; Greenfield and
Fasman,
1969) ,
These computed curves, although yielding fairly reliable
results for synthetic polypeptides, fail to account satisfactorily for the
OR D and C D spec tra
of
those globular proteins of which the structure
is known from X-ray analysis (e.g., niyoglobin and lysozyme). The
present authors feel that this type of approach is without value
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362
KEITH
J .
DORRINGTON AND CHARLES TANFORD
or justification. For example, neither myoglobin nor lysozyme has any
random coil. Nonhelical and non-&structure regions of the backbone are
not random but fixed in space. There is no reason to believe that such
regions will have optical properties comparable to randomly coiled
polypeptides. In addition, globular proteins have nonpeptide chromo-
phores (aro ma tic amino acids an d disulfide bon ds) which are known,
from model compound studies, to have complex OR D a nd C D spectra
which in many instances are highly dependent on the environment of
the chromophore. The day seems far off when the contribution of such
chromophores to the ORD and CD of a protein can be unambiguously
evaluated. Such an analysis would probably have to be supported by
X-ray data which would ren der the OR D or C D analysis redund ant . The
immunoglobulins illustrate the problems associated with the above
approach since no com bination of th e O R D or C D characteristics of t he
three conformations listed above would account for the spectra of
immunoglobulins.
T h e one stru ctu ral feature of protein molecules th at can
be
recognized
with fair certainty from ORD or CD data is the presence
of a
high
content of a-helical regions. Th e reason for this is that th e ma gnitude of
the optical activity near 230 mp., and the curvature of ORD plots at
higher wavelength,
as
represented by the
b,
parameter of the equation
of Moffitt an d Yang (1 95 6) , are m uch greater for a-helical polypep tides
than for synthetic polypeptides in other conformations. Quantitatively
similar results have been obtained for every protein known to have a
high a-helix content on the basis of other information,
but
in no case for
any protein known not to have a predominantly helical structure. These
features, therefore, seem to be specific indicators of the a-helical con-
formation.
On the basis of the experimental results, it seems fairly certain that
imm unoglobulins do not have significant amoun ts of a-helix. T he param -
eter,
b,,,
of the Moffitt-Yang equation, consistently has zero or slightly
positive values for immunoglobulins. The maximum magnitude of levoro-
tation near 230 mp.
is
only 1200-1800 deg.cma2/decimole,whereas, for
the polypeptides in 100% elical form,
[rn’],,,,,,,.
has a value near -15,000
deg.cni.‘/ decimole. Also helical p olyp eptide s have
a
maximum at 198
m p , whereas immunoglobulins have a minimum at the same wavelength.
The peptide bond absorption of yG does show marked hypochromicity
in the 190-205-mp. region (Gould et al., 1964; Ross and Jirgensons, 1968)
as has been shown for a-helical polypep tides (Im ah or i and T anaka, 1959;
Rosenheck and Doty, 1961). However, Gould et a). attribute this
decreased absorption to vicinal effects of the side-chain chroniophores or
to th e existence of somc periodic struc ture distinct from th e a-helix.
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MOLEC U LA R
SIZE
AND CONFORMATION
OF IMMUNOGLOBULINS
363
c.
P R O P E R T I E S OF SEPARATED HEAVY AND LIGHTHAINS
There is now good evidcwce that isolatcd heavy chains from
yG
anti-
body can bind homologous antigen or hapten (Fleischman et
al., 1963;
Metzgcr and Singer, 1963; Utsumi and Karusli, 1964; Haber and Richards,
1966). The equilibrium constaut for combination with antigen or hapten
is, however, usually one or two orders of magnitude smaller than that
of
each binding site of the whole molecule or of the Fab fragment. In some
instances, light chains have also been shown to
possess
small, but
measurable affinity for homologous antigen (Goodman and Donch,
1965;
Mangalo et
nl.,
1966; Yo0
et (&l.,
1967) . There are two possible explana-
tions for the observation of specific affinity with reduced binding constant
-one being that the binding site of the native antibody involves portions
of both light and heavy chains, the other Being that only one chain carries
the binding site but that the other chain is required to maintain it in a
reactive conformation. Decision between these possibilities would con-
tribute significantly to an understanding of the generation of antibody
specificity, and this has, therefore, stimulated interest in the conforma-
tional properties of the isolated heavy and light chains.
In order to separate the heavy and light chains, it is first necessary to
break the interchain disulfide bonds and
to
protect the resulting
SH
groups against reoxidation. This can bc done uiider relatively mild
conditions, but does not significantly affect the cohesion between the
chains, which is primarily due to noiicovalent interactions. Quite drastic
conditions are required to break thew interactions:
1
hl
propionic acid is
needed for rabbit yG, though 1 M acetic acid or very low pH alone will
suffice for human yG. The chains can bc separated by gel filtration in the
dissociating media and then returned to more benign conditions for
examination. The heavy chains tend to form aggregates or even to
precipitate from solution, but stable preparations can be maintained at
pH 5.5
at low protein concentrations
if
optimal procedures are followed.
Appropriate procedures for rabbit heavy chains are described
by
Bjork
and Tanford
(1970)
and for human chains by Steveiisoii and Dorrington
(1 9 7 0 ) . Even under the best conditions, aggregates tend to form slowly
and must be removed from solutions that have been stored for an
appreciable length of time. The requirements for obtaining stable prepa-
rations
of
light chains are
less
stringent than for hcavy chains.
Bjork and Tanford (1970) have shown that rabbit heavy chains have
a molecular weight of 108,000 to 117,000 by sedimentation equilibrium,
i.e.,
they exist as diniers
( H A ) .
hey are dissociated to monomers at low
pH or in 6
M
guanidine hydrochloride, showing that they were held
together b y noncovalent forces
as
is to be rxpectecl from the fact that
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MOLECULAR SIZE AND CONFORMATION
OF IMMUNOGLOBULINS
365
separated chains an d the parcnt molecule-the mag nitude of rotation was
much more negative and the characteristic Cotton effect at 240 mp. was
no longer evident. More recent studies have shown that such drastic
changes in the ORD ar e not seen if sta ble preparations fre ed of aggre-
gated m aterial are examined. Figu re 12 shows results ob tained ind e-
Wavelength (mpl
2
220 230 240 250 260
Wavelength
( m p )
FIG. 12.
The optical rotatory dispersion spectra, between 220 and 270 mG.,
and yG and of heavy- and light-chain dimers derived from it following mild reduc-
tion and alkylation. The soIvent is acetate buffer, pH 5.5. Portion A represents a
human myeloma protein
(
Stevenson and Dorrington, 1970); portion B represents
pooled rabbit yG (Bjork and Tanforcl, 1970).
pendently in two laboratories for preparations consisting of essentially
pu re H, and pure L,, f rom human and rabbi t yG. They show that
changes in conformation have accompanied chain separation.
If
no con-
formational change had occurred, the residue rotation for the native
protein would be a weighted average of the residue rotations of the two
chains, i.e., with the relative weights of
H
a n d L chains
[ N ~ ’ ] - , G =
0 . 6 8 [ m ’ ] ~
0.32[’m’]L
(1)
This relation clearly does not hold true.
(The corresponding relat ion
does hold tru e for the
ORD
curves of
rG,
Fab
and
Fc,
shown in Fig. 11.)
Although Fig. 12 thus indicates that conformational changes ac-
company chain separation, the distinctive features of the dispersion
curve a re preserved. Th e 225-mp, trough of yG appears in the ORD curve
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366
KEITH J . DORRINGTON
AND
CHARLES TANFORD
for L.,, th e 230-mp. trough an d t he 240-mp. Cotton effect are seen in the
curve for H,. It can be concluded that substantial regions of the ordered
structure of H and
L
chains in
yG
are preserved in the isolated chains,
in the dimeric form in which they a re present a t p H 5.5.
T he ORD patterns of H and
L
chains under conditions where chain
sepa ration occurs, i.e., at low p H o r in propionic acid, are, of c ourse,
quite different. The polypeptide chains are in
a
denatured state under
these conditions.
Since it is not possible to assign any feature of the
ORD
pattern
uniquely to those portions of the polypeptide chains that constitute the
binding site for antigen, these experiments cannot resolve the question
posed at the beginning of this section regarding the underlying cause for
the diminution in binding constant for antigen that accompanies chain
dissociation. Additional evidence concerning this question is, however,
provided by the recombination experiments described in Section IV,C.
IV.
Recovery of Native Conformation Following
Chain Dissociation and Unfolding
Basically, two types of experiment have been performed on t he
unfolding of immunoglobulins, almost exclusively with yG-globulin. In
the first type of experiment,
yG
is completely unfolded to the random-
coil form in which state it loses all its biological and antigenic properties.
Recovery of some specific property (e.g., antibody activity) is assessed
following refolding of the molecule. Such experiments have been used
to determine whether or not all the information required to generate a
specific three-dimensional structure resides in the amino acid sequence.
The second type of experiment aims principally at an understanding of
the interaction between the constituent polypeptide chains, e.g., the
chains are separated and hybrid molecules with chains from functionally
different parent molecules are studied. Since chain separation cannot
be achieved without partial unfolding of the internal structure of each
chain, these experiments necessarily also involve reversible disruption
of the three-dimensional structure, but not to the same extent as when a
random coil is formed.
A. REVERSIBLEANDOM-COIL
ORMATION
Buckley et al. (1963) unfolded Fab fragments, derived from rabbit
antibovine serum albumin (anti-BSA) antibody, with
6
M guanidine
hydrochloride. Under such solvent conditions, no noncovalent bonds
remain and the only restrictions
on
random-coil formation are those
from the intact disulfide bonds. (For review, see Tanford, 1969.) T h e
unfolding of F ab could b e reversed by t he slow removal of th e guanidine
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MOLECULAR
SIZE
AND CONFORMATION OF IMMUNOGLOBULINS
367
hydrochloride by dialysis. The combining ability of the native antibody
fragm ent an d th e refolded fragment was assessed b y sedimentation
velocity studies in th e presence of antigen (B S A ). Seventy-five pe r ce nt
of the combining ability of the native Fab was recovered. Physical
studies clearly showed that the native and refolded fragments were
indistinguishable.
Noelken and Tanford (1964) carried out essentially similar studies
with Fab fragments from high-affinity DNP antibody. This system has
the advantage that a sensitive fluorescence quenching technique can be
used to assess antibody activity. Approximately 70% of th e na tive F a b
activity was recovered upon refolding of protein unfolded in 6.3
M
guanidine hydrochloride.
Haber (1964) was the first to report a successful recovery of specific
antibody activity upon refolding and reoxidation of completely reduced
and unfolded Fab fragment, derived from rabbit antiribonuclease. The
Fab
fragments were unfolded in the presence of 2-mercaptoethanol in
8
or 10
M
urea or 6 M guanidine hydrochloride. The protein was ran-
domly coiled in the latter two solvents
as
judged by ORD, but consider-
able residual ordered structure
was
present in 8 M urea. Refolding and
reoxidation ( i n a ir ) of F a b was achieved by dialysis, first, again st low
concentration
( l o -”
M )
of
buffered 2-mercaptoethanol ( p H
8)
and,
second, against buffer alone at p H 8. Under optimal conditions, 20-27%
of the original binding activity, as measured in
a
radioimmunoassay
system, was recovered following refolding and reoxidation of protein
reduced in 10 M urea or
6 h l
guanidine hydrochloride, or 568 from
protein reduced in
8
M urea.
Whitney and Tanford (1965a) performed similar experiments on
anti-DNP Fab fragments unfolded in 6 M guanidine hydrochloride and
reduced with 0.1
M
2-mercaptoethanol. Oxidation and refolding of the
protein resulted in the recovery of 14 to 24% of th e original activity as
judged by fluorescence quenching. Reoxidation in the presence of excess
antigen enhanced the amount of activity recovered to
a
small extent.
In a sepa rate study, Wh itney an d Tanford (1 96 5b ) assessed how far the
reoxidized Fab was comparable to the native protein using several
physical techniques. An tigenic analysis, sedimentation velocity, an d O R D
studies indicated that the reoxidized protein was very similar to native
Fab. The principal difference was seen in the ORD where the Cotton
effectnear 240 mp,, clearly seen in native Fab, is only partially recovered
in the reoxidation product.
Freedman and Sela ( 1966a,b) have reoxidized completely reduced
and unfolded rabbit
yG
and assessed the recovery of both antibody
activity and antigenic determinants. They utilized an earlier finding
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368
KEITH J . DORRINGTON A N D CHARLES
TANFORD
(Fuch s and Se la, 1965) that the at tachment of ~ ~ - a l a n i n eeptides to
YG enhance s its solubility proper ties to t he ex tent of yielding a soluble
molecule even when completely reduced. Initial studies on the recovery
of antigenic determinants were performed
on
nonspecific poly-DL-alanine
yG (Freedman and Sela, 1966a) using a goat antiserum against rabbit
nonalanylated
yG.
I t ha d been demonstrated previously tha t th e poly-
DL-alanylation did not significantly affect the antigenic structure of yG
(Fu ch s and Sela, 1965) and th e use of nonalanylated antigen in th e goat
avoided the production of antibodies against th e DL-alanine peptides. T h e
poly-DL-alanine yG was r ed uc ed with 2-mercaptoethanol eith er in 8 to
1 0 M urea or in 6 to 8 M guanidine hydrochloride. Optmial conditions
for reoxidation were determined, and these allowed recovery
of
almost
all the antigenic determinants, as judged by quantitative precipitin
analysis, although up to
4
times more reoxidized protein was required.
Since the antigenic determinants on
yG
are predominantly conforma-
tional ( a s opposed to se qu en tial) , recovery of an tigenic activity following
reoxidation is a good index of the recovery of the native conformation.
As 95-100%
of
the reoxidized poly-DL-alanine yG was soluble at pH 8,
compared with 5 to 10% of u nsubstituted yG, recovery
of
antigenic
activity could be assessed in essentially all the molecules. It was found
that the recovery
of
the antigenic determinants was more complete and
the efficiency of precipitation was greater when the heavy and light
chains were first reoxidized separately, suggesting that the reoxidation
process involves the initial formation of intrachain disulfide bonds fol-
lowed by the formation of interchain disulfides. This observation is
consistent with the current experimental evidence on the biosynthesis
of immunoglobulins.
In their second paper, Freedman and Sela (1966b) s tudied the re-
covery of specific antibody activity following reoxidation of completeIy
red uc ed rab bit poly-DL-alanine anti-BSA. Th e immunospecifically purified
poly-DL-alanine antibody was reduced with 2-mercaptoethanol in 8 M
guanidine hydrochloride. Upon reoxidation, 25% of th e antigen-binding
activity was recovered in an assay measuring I3'I-BSA-binding capacity.
A second assay system involving the inhibition of th e homologous BSA-
anti-BSA precipitin reaction indicated a 50%recovery of activity. The
difference in the estimates of antibody activity recovered was probably a
function of the incomplete recovery of the antigenic determinants
of
t he
poly-DL-alanine 7G. T he 1311-BSA-binding ssay de pends on t h e pre cipita-
tion
of
th e antibody-antigen complex with goat anti-yG, whe reas the
inhibition reaction does not involve this type of interaction. The reoxida-
tion conditions giving maximum recovery of antigenic determinants did
not yield optimal recovery of antibody activity, Freedman and Sela
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MOLECULAR SIZE AND
CONFORMATION
OF
IMMUNOGLOBULINS
369
(
1966b) interpreted this as indicating that re-formation of the combining
site is an intrachain event, whereas recovery of antigenic determinallts
involved more than one polypeptide chain. This conclusion was supported
by the observation that the antibody activity recovered was independent
of whether the heavy and light chains were reoxidized separately or
together, in contrast to their findings regarding the recovery of antigenic
determinants.
In a recent report from Sela's group (Jaton
e t
ul., 19 68), recovery of
specific antibody activity upon reoxidation
of
completely reduced heavy
chain a nd F d fragmen t has been studied. Poly-DL-alanine heavy chain
and poly-m-alanine Fd were prepared by mild reduction of PO~Y-DL-
alanine anti-DNP and cyanogen bromide cleavage of the poly-DL-alanine
heavy chain, respectively. The polyalanylated heavy chain and Fd were
soluble in aqueous solvents at neutral p H (F uc hs and Sela, 1965) and
possessed an average of 0.26 and 0.16 hapten binding site, respectively.
The average association constants
( K , )
for thcse sites on both proteins
was
2.4 X l o G
M - ' , approximately two orders of magnitude lower than
the intact antibody. Complete reduction was achieved with
0.4
M 2-mer-
captoethanol in 8 M guanidine hydrochloride. Following reoxidation, 37%
of the hapten binding sites on the poly-DL-alanine heavy chain were
recovered, a nd 59% of those
on
the alanylated Fd. The
K ,
for the re-
formed sites on the heavy chains was
1.3x 10';
M-' and 0.6 X 10" M-' for
the Fd fragment, i .e., the same order of magnitude as before reduction.
These resuIts support the earlier concIusions of Freedman and Scla
(1966b) that the formation of the binding site per
se
is an intrachain
event since recovery of binding activity can occur in the absence of the
light chain. Further, formation of the site requires only a portion of the
heavy chain ( F d ) . This does not, however, invalidate other evidence
that the light chain has an, as yet, undetermined role in influencing the
binding characteristics of th e combining site (S ection
I V , C ) .
The results
of
the experiments described above have bearing on two
important areas of protein chemistry:
1. I t has been proposed, principally b y Anfinscn (1962, 19 67 ), tha t
th e folding an d cross-linking of polypep tide chains occurs spontaneously,
directed solely by thermodynamic forces dependent on the amino acid
sequence alone. This concept fits well current observations on the syn-
thesis of proteins which provide for a transfer of sequential information
from the base triplets of the nucleic acids to the amino acids of the
protein without provision fo r th e transfer of conformational information.
Experimental verification of Anfinsen's hypothesis has been obtained with
a
nu m be r of single-chain proteins ( f o r references, see Neum ann
et
ul.,
1967). However, recovery of biological activity following complete
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370
KEITH
J . DORRINGTON AND CHARLES TANFORD
reduction of protcins with m ore tha n one polyp eptide chain ha s been less
convincing. The results with Fab and intact yC, clcarly show that levels
of activity significantly greater than expected from random recombination
can be achieved with multichain proteins.
2. Broadly speaking, theories of antibody production fall into two
principal categories ( Burn et, 19 69 ). Selective theories propose th at ea ch
potential antibody-producing cell is programmed to make only a single
type of immunoglobulin with
a
defined sequence. T he a ntigen selects, by
some poorly understood mechanism, the cell or cells able to produce an
antibody showing good binding characteristics for itself. Proliferation
subsequently takes place to form
a
clone of antibody-producing cells.
The
antigen does not induce major, irreversible conformational changes
in the antibody-combining site.
In the second type of theory the conformation of the antibody-
combining site is controlled by t h e antigen d ur ing biosynthesis of the
immunoglobulin.
In
other words the antibody molecule is adapted to fit
the particular antigenic determinant. The adaptive conformational change
remains stable while the molecule is in its native state but is lost on
unfolding. Recovery of the binding site would, therefore, require the
presence of the antigen
during refolding. The reoxidation-refolding
experiments are compa tible only with
a
selective mechanism since specific
binding sites are formed in the absence of antigen. Further, in experi-
ments in which reduced nonspecific yG (o r F a b ) has been reoxidized in
the presence of antigen, no evidence of induced binding ability could
be detected (W hitne y and Tanford, 1965a).
B. REVERSIBLE
DISSOCIATIONNTO
HALF-MOLECULES
Rabbit
y
Gcan
be
dissociated into half-molecules (i.e., on e heavy an d
one light chain ) by reduction of the single inter-heavy-chain disulfide
bond and disruption of the noncovalent interactions between the Fc
portions by lowering the pH to 2.5. The process can be carried out
without alteration in the Fab portion of the molecuIe, and, since the
antibody-binding site is contained wholly within the Fab portion, recom-
bined half-molecules are indistinguishable from native antibody ( Palmer
et
al., 1963; Palmer a n d Nisonoff, 19 64 ). Hyb rid molecules co ntaining two
binding sites with different specificities are readily obtained ( Nisonoff
and Hong, 1964).
These experiments constitute control experiments for those described
in the following section. The corresponding experiments cannot b e carried
out with human YG because disruption of the noncovalent interactions
between the Fc portions of human heavy chains occurs simultaneously
with disruption of the Fab portions of the molecule.
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MOLECULAR SIZE
AND
CONFORMATION
OF
I M M U N O G L O B U L I N S 371
C.
DISSOCIATIONN-m H E A V YN D LIGHT
CHAINS
ND
ITS
REVERSAL
This section is concerned with reversal of the lesser perturbations in
conformation that accompany dissociation of mildly reduced yG to
heavy and light chains at low pH or in propionic or acetic acid solution.
I t was noted earlier that the separated chains may be returned to p H
5.5 and that they exist there as dimers ( H L a nd L 2 ) , which retain many
of the conformational features seen in yG. However, there are also con-
formational differences, notably a close association betwee n th e F d
portions of the two heavy chains, which appears to have replaced the
close association between Fd and
L
chains in native yG. In the experi-
ments described here, dissociated and separated H and L chains are
mixed in equimolar proportiom, and dialyzed to nearly neutral
pH
in
an aqueous medium, or, alternatively, a mixture containing separated
chains is return ed to nearly neutral p H w ithout actual fractionation into
solutions containing only H and only L chains.
It has been demonstrated that reiiaturation of this type leads to re-
formation of a 7 S molecule (Edelman
et al.,
1963; Olins and Edelman,
1964; Roholt
et
al., 1964) , although the extent of recombination shows
considerable variation among proteins
(
Gordon and Cohen,
1966) .
T he
recombined molecules, their subunits held together by noncovalent forces
alone, resemble the original immunoglobulin as judged by sedimenta-
tion velocity, molecular weight, antigenic structure, and electrophoretic
properties. Recombination can be achieved between chains derived from
different classes of immunoglobulin (Gally and Edelman, 1964; Grey
an d Mannik, 1965) and different species (Fougereau et al., 1964) .
Significant recombination of heavy and light chains does not occur
when the chains are mixed at neutral pH, under which conditions light
chains exist
as
dimers and heavy chains
as
dimers and insoluble aggre-
gates. Th e ap pa ren t requirem ent for mixing of th e chains in organ ic acid
solution at low pH prior to recombination has been interpreted to mean
that monomeric chains are the species involved in the recombination
reaction. (B ot h heavy a nd light chains are principally m onomers a t
pH
2.5.)
However, Stevenson has shown that rapid recombination of heavy
and light chain dimers occurs at pH 5.5 (Stevenson, 1968; Stevenson and
Dorrington, 1970) , the overall reaction being y L +L1+- 2L2. t
is
likely
that the mechanism involves prior dksociation of L, to monomeric L
chains, i.e., L, + L, L
+
y 2+ LyI, Lyl
+
L -+LyyL. Disulfide-bonded
dimers of light chains do not combine ‘It all with y. .
The most interesting aspect
of
the recombination process is its
specificity. M easuremen ts of affinity betw een antige n a nd antibody have
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372
KEITH
J .
DORRINGTON AND CHARLES
TANFORD
always demonstrated requirement for a high degree of specificity. The
heavy chain of an antibody, in the absence of light chain, generally
combines with specific antigen, but with an affinity much less than that
of th e native antibod y molecule o r its F a b fragment. Recom bination wit h
L chain invariably results in marked enhancement of the affinity only if
homologous light chains ar e used. H eterologous light chains lead to little
if any enhancement (Edelman
e t
al., 1963; Franek and Nezlin, 1963;
Metzger and Mannik, 1964; Roholt e t
at.,
1964, 1965a,b; Hong and
Nisonoff, 1966; Lamm et al., 1966; H ab er an d R ichards, 19 66). Maximal
recovery of activity in recombined yG requires that the light chains are
derived from the same animal as well as from antibody of the same
specificity (Roholt
e t
al.,
19 65 a,b ). Zappacosta an d Nisonoff
(
1968) have
shown however th at th e binding activity of rab bit anti-DNP h eavy chain
was significantly enhanced by light chains from antibody of the same
specificity raised in other rabb its of th e same allotype. H on g a n d Nisonoff
(1966) showed that when anti-DNP antibody from a single rabbit was
fractionated into populations of high and low binding affinity, preferen-
tial enhancement was exhibited by light chain from the same fraction as
th e heavy chain.
Further evidence for specificity in the association of heavy and light
chains has come from studies with myeloma proteins. Grey and Mannik
(1 96 5) showed, using I3lI-labeled light chains, th at th e heavy chain of a
yG myeloma protein preferentially recombined with its autologous light
chain in the presence of heterologous light chain. However, considerable
variation was exhibited among light chains from different myeloma
proteins in their ability to replace autologous light chains. The competi-
tion between various light chains for a particular heavy chain has been
studied in more d etail by Mannik (19 67 ). Th e preferential recombination
of autologous heavy an d light chains was confirmed bu t seem ed to be
variable among different myeloma proteins. In some instances, preferen-
tial recombination could still be demonstrated in the presence of eighty-
fold excess of heterologous light chain. As the proportion of heterologous
light chain increased the preferential recombination decreased. Normal
yG light chains were shown to contain populations of molecules that
could replace autologous myeloma light chains in recombination with
their heavy chains. The ability of heterologous light chains to substitute
for autologous light chain was not related to either antigenic type ( K or
h )
or electrophoretic mobility
of
the parent protein or to the banding of
isolated light chains in alkaline starch-gel electrophoresis. Roholt et al.
(
1967) have devised a convincing demonstration of the preferential
recombination of antibody subunits. They prepared heavy and light
chains from two high affinity rabbit antihapten antibodies and made
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MOLECULAR SIZE AND CONFORMATION OF IMMUNOGLOBULINS 373
heterologous recombinants which showed little or no antibody activity.
Both types of recombinant were mixed in propionic acid and dialyzed to
neutrality to re-form
7
S
molecules. When assayed for the original anti-
hapten activity the molecules showed activities much higher than would
be anticipated as a result of the random re-formation of specific sites.
These observations tend to suggest that noncovalent interaction between
heavy and light chains involves the variable region of the light chain.
Ruffilli and Givol (1967) have presented some evidence that both the
variable a nd constant halves of light chain ar e involved in the interaction
with heavy chain.
The integrity of the conformation of reformed yG has been tested by
ORD
measurements, and the results at first appeared to provide an in-
triguing parallel with antibody activity measurements, i.e., full restoration
of t he ORD spectrum of native yG occurred only when heavy chains
were recombined with autologous light chains ( Dorrington et al.,
1967).
These results, and speculations about the role of H-L interactions in the
generation of antibody specificity based on them (Tanford,
1968),
have
now been shown to be at least partly incorrect. Improvements in tech-
nique which led to isolation of heavy chains with much less loss
of
characteristic conformations1 features tha n in earlier experiments ( Sec-
2
220 230 240 250 260
Wavelength (rnp)
FIG.
13.
The optical rotatory dispersion spectrum
(ORD) of
purified, recom-
bined, human myeloma yC compared with the native, untreated protein between
220
and
270 m p .
The third curve represents the
ORD
spectrum for an equimolar
mixture of heavy and light chains, c;ilcidated according to Eq. 1
).
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374 KEITH J. DORRINGTON AND CHARLES TANFORD
tion II1,C) have also led to the conclusion that full restoration of the
native ORD spectrum may not require that a heavy chain be combined
with
a
uniquely matching light chain.
Figure 13 (Dorring ton a nd Stevenson,
1970)
shows the ORD curves
of a native human myeloma yG, the weighted average of the ORD
curves of the separated chains of the sam e protein, calculated according
to Eq. ( l ) , nd the ORD curve for the reconstituted protein. The
latter is clearly identical, within experimental error, with the curve for
the native protein, showing that no irreversible conformational change
accompanies chain separation when the H and L chains are autologous.
Figure
14
(Dorrington and Stevenson,
1970)
shows similar results for
pooled normal human
yG.
n this case the separated chains must con-
stitute a highly heterogeneous mixture, and the concentration of auto-
logous light chains for a given heavy chain must be very small, so that
the bulk of the recombined molecules must contain heterologous chains.
T he yield of 7 S y G was not 100%n this experiment, some unrecombined
chains remaining in the mixture. They were removed before the ORD
measurements were made,
so
that the results reflect the properties of the
reconstituted protein alone. It is seen that the native ORD spctrum is
substantially, but not fully recovered. However, similar experiments
2o
2Ao
d o 2Ao A 0
Wavelength m p)
FIG. 14.
The optical rotatory dispersion spectrum
( O R D ) of
purified, recom-
bined, pooled, nornial human yG compared with the native, mildly reduced and
alkylated protein, between 220 and
270
nip. The third curve represents a calculated
ORD spectrum for an equiinolar mixture of heavy
and
light chains froin the same
yC.
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MOLECULAR SIZE AND CONFORhlATION OF 1h.IMUNOGLOBULINS
375
with pooled normal rabbit
yG
(Bjork and Tanford, 1970) have yielded
a product (about 75%yield) which was indistinguishable from native
yG
by ORD measurements
as
well
as
by other techniques. T h e reason
for th e discrepancy between the two results has not yet been established.
It is evident, in any event, that heterologous H-L combinations can
differ only minimally in conformation from autologous combinations.
Taken at face value, these experiments suggest that the failure to
obtain full biological activity when the
H
chain of an antibody is com-
bined with
a
heterologous
L
chain is not d u e to failure to achieve restora-
tion of conformation. The possibility exists that there are important con-
formational features which do not contribute significantly to the ORD
pattern in the wavelength range examined, and it is obviously necessary
to examine the conformational integrity of the renatured yG by other
methods. However, if other methods yield similar results, it must be con-
cluded that the H-chain conformation in the reconstituted
yG
is inde-
pendent of the nature of the L chain with which it is combined, and
in that event the effect of the nature of the L chain on the specific
affinity for antigen can only be interpreted
as
indicating that portions
of both chains form part of thc antibody-binding site.
Even this conclusion must be regarded as very tentative, however.
The foregoing results have shown that the conformation of recombined
yG ( a nd ,
as
shown in Section III ,C, that of the th e separate d
H
chains)
is very sensitive to the exact experimental procedure used to separate
and recombine the polypeptide chains. To be certain that H a nd L com-
binations exist which appear to have
both
full conformational integrity
and diminished affinity for specific antigen, it is necessary to monitor
conformation and activity on the same sample, This has not been done
to date.
V.
Conc lus ions
It is apparent from the work described in this review that we are
still a long way from
a
complete analysis of the structure of immuno-
globulins in three-dimensional terms. W e are, however, only
a
little over
a decade away from the first realization that immunoglobulins are
multichain proteins. Since that time tremendous advances have been
made in the sequence analysis of these biologically essential molecules.
One suspects that correlations of amino acid sequence and antibody
specificity are just around the corner. In addition, we are able to propose
topographical models, at least for
yG
and y M , with a reasonable degree
of
confidence. Refining these models to the atomic level still seems a
long way off. T he reasonable, assumption is that such knowledge can
come'
only from X-ray crystallography. A s incntioned earlier the first
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376
KEITH J . DORRINGTON AND CHARLES TANFORD
steps in this direction have already been taken. The bulk of the time-
consuming work, such as the preparation of suitable heavy metal iso-
morphous replacements, has still to be attacked. With appropriate com-
mitments of resources and manpower we could have an atomic picture
of an
antibody or at least an antibody Fab fragment before the end of
the decade.
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AUTHOR
INDEX
Numbers in italics refer to the pages on which the complete references are listed.
A
Abbot, A., 289, 293, 298, 301, 302, 328
216, 217, 220, 223, 224, 227, 228,
246, 247, 252, 255, 258, 266
Abel,
C.
A., 79,
83,
108, 339, 376, 378
Abeyounis,
C.
J.,
182,
191
Ada,
G.
L., 100, 106, 108, 109, 114, 224,
246, 258, 277, 281, 287, 288, 289,
290, 292, 293, 295, 298, 299, 300,
322, 328, 331
Adachi, M., 108
Adams, W. E., 136, 200
Adamson,
I.
Y. R., 212, 259
Adee,
R. R.,
292, 294, 329
Adler, F. L., 173, 191, 218, 258, 261,
266,
271, 275,
279,
287, 296, 304,
322, 325, 329
Agarossi, G., 209, 232, 258, 260
Agnew, H.
D.,
226, 263
Aikin, B. S., 305, 311, 323
Al-Askari, S., 163,
191
Albertini, R.
J.,
255, 256, 257, 258
Albright, J. F., 202, 203, 213, 214, 231,
264, 271, 281
Albritton,
W .
L., 87, 108
Alescio-Zonta, L., 6, 53
Ali,
S .
Y.,
305, 310,
322
Allegretti,
N.,
174, 194
Allen, E. M., 205, 261
Allen, H. D., 255, 256, 261
Allen, J. C., 11, 53
Allen,
J. M.,
298, 300, 301,
322
dlfrey, V. G., 317, 319, 320, 322, 327,
Alling, D., 179, 191
Allison,
A. C.,
316, 317, 322
Almeida,
J.,
70,
108
Alonso,
R.,
60, 109
Amaduo,
L.,
254, 261
Amante,
L.,
99, 101,
114
Ambrose, C. T., 276, 279
Amiel, J. L., 256, 264
T., 96,
Abdou,
N.
I,,
207,
210, 211, 214, 215,
A1’los~ D. B.,
118,
15@,68, 1799
182, 183,191, 199,200
233, 235, B 6 , 237, 239, 240, 245,
Anderson J. L., 255, 256, 257, 258
Anderson, N. A., 173, 200
Anderson,
R.
E.,
176,
200
Anderson, ’.
G.,
6oy
Anderson, B., 216, 220, 230, 231, 246,
Andre,
J. A.,
163, 191
Andreevn, N. 94, 109
Anfinsen,
C. B.,
369, 376
Anteunis, A,, 317, 329
Anton, E., 317,
323
Archer,
A.
G., 307, 322
Archer,
G.
T.,
306,
329
Argyris,
B. F.,
218, 219, 221, 248, 254,
258, 285, 322
Armstrong,
B.
A., 293, 298, 322
Armstrong, P., 191
Armstrong, W. D., 206, 225, 228, 252,
Arnason,
B. C.,
250,
263,
272,
280
Aronow, D., 293, 296,322
Aronson,
A.,
293, 296, 322
Aronson,
hl.,
244,
258
Aronson, N. N., 322
Arquilla, C .
R.,
175,
191
Artenstein, M.
S.,
48, 49, 54
Asherson, G. L., 173, 191
Ashley,
H.,
276, 279
Ashman,
R.
F., 60, 65,
90,
107, 108
Ashwell, G., 73,
I13
Askonas, B. A., 5, 38, 53, 67, 68, 69, 70,
75, 100, 102, 114, 218, 219,
258,
269, 287, 288, 289, 290, 293, 295,
300, 303, 304,
322, 326, 327,
330,
355, 380
N. G., 156,
269
F , 283, 305, 324
258, 273, 279
329
Asofsky,
R.,
202, 228, 231, 267
Aspinall,
R.
L., 272, 279
Astorga, G., 313, 322
383
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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384 AUTHOR INDEX
Astrin, K., 50, 51, 53
Atkinson, J. B., 256, 258
Atsumi,
T.,
108
Attardi, G., 229, 258
Auer, G., 320, 331
Auerbach, R., 242, 243, 262, 269
August,
C.
S., 272, 278, 279, 280
Aust, J. B., 172, 195
Aust,
J. C.,
256, 265
Austen, K . F., 40, 54, 308, 309, 326, 328
Austin, C. M., 100, 114, 237, 254, 258,
Auzins, I., 288, 293, 295, 322, 326
Avery,
0.
T.,
32,
53
Awdeh, Z. L., 5, S3
Axelrod, M., 220, 263
Axelsson, H., 376
Axen, R., 33, 53, 56
265, 288, 289, 290, 322
B
Bach, F. H., 179, 191, 255, 256, 257, 258
Baddiley, J., 46, 53
Baehner,
R.
L., 313, 322
Baggiolini,
M.,
307,
322
Baglioni,
C.,
6, 53, 349, 376
Baillif, R.
N.,
288, 322
Bain, B., 179, 191, 192, 207, 258
Bainbridge, D. R., 171, 192
Bainbridge,
J.,
161, 192
Bainton, D. F., 306, 307, 330
Bairati, H., 290,
329
Balner, H., 211, 220, 258
Bangham,
A. D.,
9 3 , 1 0 8
Barge,
A.,
150, 152, 193
Barker, B.
E.,
316,
324
Barnes,
D. W.
H.,
208, 212, 234, 255,
257, 258,261
Barnhart, M., 305, 329
Barrett, A.
J.,
305, 310, 322, 330
Barry, C. B., 91, 114
Barth, W. F., 101, 108, 225, 261
Bartin, M. M., 255, 256, 257, 258
Bastos, A. L., 317, 322
Batchelor, J. R., 119, 129, 148, 168, 187,
Battisto, J. R., 251, 259
Baudhuin, P., 292, 294, 330
Bauer, D. C., 92, 109, 355, 376, 379
Bauer, J. A., 175, 192
Baumann, J. B., 168, 200
192, 199
Beale,
D.,
62, 63, 74, 79, 80, 82, 83, 84,
86, 108, 355, 376
Bean,
M.
A., 306,
327
Beckwith, J., 58,
115
Bedford,
D.,
206,
260
Bedford, M., 220, 244,269
Benacerraf, B., 4, 40,
55,
96, 97, 105,
108, 114, 115, 175, 189, 196, 197,
202, 213, 214, 217, 218, 229, 237,
239, 240, 250, 251, 254, 260, 261,
263, 266, 267, 276, 278, 280, 281,
285, 286, 289, 301, 323, 325, 329,
330, 372, 379
Bendinelli,
M.,
218, 219,
259
Benditt, E.
P.,
307, 327
Benezra, D., 179, 192, 207, 259
Bennet,
W.
E., 291, 322
Bennett, B., 189,
192,
316,
322
Bennett, J. C., 78, 80, 81,
87,
108, 111
Bennett,
M.,
208, 209, 213, 259, 260
Bennich,
H.,
340, 341, 349, 376, 377,
Bensch, K. G., 298, 325
Benson, B., 292, 294, 299,
323
Berg, T., 101, 112
Berggard, I., 349,
378
Berk, R.
S.,
301, 322
Berken, A,, 97,
108
Berkin, F., 292, 295, 300, 328
Bernardi, G., 305,
322
Berrian, J. H., 160, 197
Bert, G., 215, 259
Beychok,
S.,
357, 376
Bezer,
A.
E., 48, 56
Bianco,
C.,
286,
289,
322, 324
Bickis, I. J., 183, 192
Bierring, F., 208, 209, 259
Billingham, R. E., 117, 119, 136, 145,
146, 157, 159, 160, 163, 165, 170,
192, 195,
202, 248, 249, 251,
259,
269
378, 381
Binaghi, R. A., 97,
109,
217, 259
Binet, J. L., 296, 325
Bing,
D.
H., 50, 56
Biozzi,
G.,
97,
109,
206, 217,
259
Birch-Andersen, A,, 220,
266,
292, 293,
Biro,
C.
E., 95,
113,
240, 254,
259
Bishop, D. C., 302,322
Bishun, N . P.,
209, 259
295, 329
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX 385
Bitensky,
L.,
309,
324
Bjork,
I.,
349, 363, 364, 365, :375,
376
Blackburn,
W .
R.,
256,
262
Blandamer, A .
H.,
120,
192
Blanden, R. V., 202, 221, 259, 264, 285,
Blease, M., 171, 172, 197
Bloch, K. J., 12, 29, 56
Bloom, B. R., 189, 192, 316, 322
Blotli, B., 68, 70,
109, 115,
352,
376, 381
Bluhm, G. B., 305,329
BIyth,
J. L.,
247,
269
Boak, J. L., 212, 220, 259, 262
Bockman,
R .
S. ,
88,
114
Bodnier,
J.,
183, 184, 192
Bodnier,
W .,
183, 184,
192, 198
Bold, E.
J.,
183, 200
Bollett, A.
J.,
313,
322
Bond,
V.
P., 208, 210, 263, 268
Bonhag, R., 209, 225, 230, 2.31,
268
Borek, F., 275,
282
Borel, J .
F.,
305, 315,
322
Borjesan, J., 317,
324
Borsos,
T.,
64,
5, 96,
108, 109,
111
Boulund, L., 319, 324
Bouroncle, B. A., 317,
323
Bourrillon,
R.,
73,
108
Bouthillier,
Y.,
206,
259
Bowden, D.
H.,
212,
259
Bowers,
W . E.,
292, 293, 294, 299,
322
Boyce, C. R., 43,
56
Boyden,
S. V.,
220,
265
Boyle,
W.,
129,
192
Boyse, E.
A.,
128, 150, 156, 183, 186,
192, 193, 197
Bradley, T.
R.,
204,
259
Braga Andrade,
F.,
97,
109
Brandes,
D.,
317,
323
Bratt,
G. J.,
349,
381
Braun,
D.
G., 8, 9, 14, 15, 19, 22, 24, 25,
Braun, W., 162, 192, 277, 278, 281, 285,
Brdicka, R., 117,
199
Breckron,
G.,
258
Brederoo, P., 290, 294,
324
Brenncman, L., 42, 53
Brent, L., 119, 123, 136, 145, 146, 147,
157, 159, 160, 161, 163, 165, 169,
301,
327
32, 37, 38, 49, 51, 52, 53, 54
323
170, 172, 174, 176, 178, 181, 182,
FJ2, 251,
259
Brent,
V. D.,
118, 123, 148,
193
Brieger,
E.
hf., 301,
322
Briggs, D. R . , 73, 111
Briles,
D.,
33,
55
Brittinger, G., 290, 317, 318, 319, 320,
323, 326
Britton, S., 275, 279
Brock, T. D., 118,
193
Brotly, J. I . , 204, 266
Brooke, J. S., 251,
259
Brown, A., 342, 379
Brown,
F.,
70,
108
Brown, J. B., 160, 163, 164, 174, 176,
178,
192
Brown, J. R., 369, 379
Brown, R. J., 29,
55
Brown, R. S., 305, 306, 327
Brownhill,
L. E.,
316,
324
Brundish,
D.
E., 46, 53
Brunette,
D.
M.,
259
Bruning, J . W., 118, 123, 148,
193
Brunner,
K.
T.,
156,
193
Bruton, 0.C., 272, 279
Bryant, B. J., 208,
267
Brycenson, A. D. M., 316, 327
Buckley, C. E., 335, 341, 342, 343, 350,
366,
376, 379
Bum, C., 317,
329
Burger, M., 226, 227, 259
Burke, J.
S.,
306, 312,
323, 328
Burnet,
F.
M., 118, 170, 193, 246, 259,
272, 279,
282,
370,
376
Burstein,
M.,
59,
108
Burstein,
N. A,,
251,
259
Burwell,
R.
G . , 244, 267
Bush, G.
J.,
136, 170,
193, 200
Bush,
S.
T., 88,
109
Bussard,
A. E.,
205,
263,
290,
323, 326
Buttress,
N.,
62, 63, 75, 79, 80, 82, 83,
84, 86,
108, 110
Buyukozer, I., 290, 293, 295,
323
Byers,
V. S.,
231,
259
Byrt, P., 106,
108, 109,
224, 246,
258
C
Caffrey,
R.
W., 208, 261
Cain,
W .
A,, 272,
279
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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386
AUTHOR INDEX
Calcott, M. A,, 95,
113
Callaghan,
P.,
65, 109
Calvanico,
N.,
381
Cammack, K.
A.,
335, 376
Campbell, D.
H.,
275, 277, 280, 282, 285,
Campbell, P. A,, 205, 229, 263
Canning, R. E., 59, 62, 63, 71, 110
Cannon,
J.
A.,
176, 183, 200
Cannon,
J.
H., 256, 268
Capra, J. D., 7, 56, 100, 116
Carbonara,
A. O.,
76, 109
Carlton, D. A., 314, 327
Carpenter, C.
B.,
287, 323
Carpenter,
F. H., 8, 56
Carter, P. K., 176, 200
Carter R . L., 251, 260
Carter, T. C., 255, 259
Casassa, E. F., 337, 376
Casley-Smith, J. R., 291, 295, 298, 323
Castermans, A., 136, 145, 146, 193, 195,
Catanzaro, P. J., 292, 295, 302, 323
Cathou,
R.
E., 349, 358, 359, 360, 376
Catty, D., 6, 53
Cavins, J. A,, 235, 259
Cebra, J. J., 99,
100,
101, 109, 114, 254,
263,340,376
Celada, F., 161, 167, 184, 193, 206, 230,
259
Centeno,
E. R.,
40, 54
Ceppellini, R., 117, 119, 136, 146, 150,
152, 177, 184, 193, 196, 200
Cerottini, J. C., 156, 193, 220, 244, 269,
277, 279, 288, 296, 300, 302, 303,
327, 330
288, 303, 323
196
Chahin, M., 290, 328
Chamberlain, C. C., 183, 200
Chambers, L. A,, 136, 193
Chandler, J. G., 248, 267
Chaperon,
E. A.,
203, 214, 222, 227, 229,
233, 236, 237, 238, 259, 260, 271,
273,279,285,323
Chaplin, H., 59, 82, 109, 376
Chapman,
J.
A , ,
317,323
Chapman, N. D., 259
Chase, M.
W.,
175, 193, 202, 251, 259
Chavin, S . I., 90, 109
Chayen, J., 309, 324
Chen, J. P., 63, 84, 86, 109, 353, 376
Cheng, V., 209, 225, 230, 231, 268
Chernokhvostova,
E.,
94, 109
Cherry,
M.,
121, 195, 196
Chesebro, B., 59, 68, 109, 115, 352, 376,
Chessin, L. N., 317, 324
Choi, T. K., 26,
56
Chowdhury, F.
H.,
344, 376
Christie, G.
H.,
212, 220, 259, 262, 263
Church,
J.
A., 184, 194
Churchich, J., 344, 376
Chutna,
J.,
168, 177, 193
Cinader, B., 214, 259, 276, 277, 279, 282
Cioli,
D.,
6,
53, 349, 376
Claflin, A. J., 205, 259
Claman, H.
N.,
203, 214, 222, 227, 229,
233, 238, 237, 238,
259,
260, 271,
273,279, 285, 286,323
Clamp, J.
R.,
72, 73, 109, 115
Clarke, C.
M.,
208, 234, 235, 264
Clarke, D. A., 301, 330
Clarke,
J.
A., 93, 114
Clausen, K. P., 317, 323
Clem,
L.
W.,
88,
102, 103, 104,109,112,
114, 356, 376
Cline, M. J., 305, 309, 328
Clondman, A. M., 162,199
Cochrane,
C .
G., 96, 109, 116, 202, 203,
228, 260, 304, 305, 309, 311, 313,
315,323, 329
Cohen, E. P., 218, 265, 271, 279
Cohen, M.
W.,
248,260
Cohen,
S.,
2, 53, 59, 75, 77, 82, 83, 101,
109,
111,
250, 260, 289, 323, 333,
371,376, 377
381
Cohn, E. J., 337, 376
Cohn, M., 44, 46, 53, 56, 99, 109, 229,
258, 333, 338,379
Cohn, Z. A., 220, 221, 222, 260, 269, 288,
290, 291, 292, 293, 294, 295, 299,
300, 301, 303, 322, 323, 324
Colberg, J. E., 100, 109
Cole,
L.
J., 205, 206, 208, 209, 229, 238,
Cole, N. H., 96,
109
Cole, R. M., 300, 325
Coligan, J. E., 92, 109, 355, 376
Colombani,
J.,
128, 150, 152, 183, 193,
Colten, H.
R.,
95, 98, 109,
114
249,
255, 262, 265, 269
194
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX 387
Condie,
R. M., 380
Congdon, C. C., 210, 226, 255, 260, 261:
Conger,
A.
D., 226, 261
Connell,
M. J.,
205, 206, 230,
267
Converse, J .
M.,
119, 135, 160, 198
Coombs,
R.
R. A., 206, 215, 217,
260,
Coons, A.
H.,
203, 213, 214, 231, 251,
Cooper,
A.
G., 77, 90,
109, 110
Cooper, M. D., 202, 226, 260, 261, 272,
279, 280
Coppleson,
L.
W.,
214,
265,
271,
281,
286,
328
Cornely, H. P., 315,
323
Corsi,
A.,
226,
260
Corson, J.
M.,
165, 172,
193, 197,
198
Cosgrove, G.
E.,
235, 255,
266
Costea,
N.,
87, 92, 101,
109, 114,
116
Cottier, H., 208, 263
Coultier,
M .
P., 255,
266
Counts, R. B., 42, 55
Cowdrey, C.
R.,
212, 220,
266
Cowling, D. C., 207, 260
Crabbir,
P. A. , 378
Craddock, C. G., 242, 260
Craig, L. C., 25,
54,
88,
114
Crandall, M .
A.,
118, 193
Cree,
I.
C., 256,
265
Crittenden, L. B., 117, 193
Cross,
A. M.,
202,
260, 264
Crowder, J. G., 307, 313, 323
Crowle, A.
J.,
174,
193
Cruchaud,
A.,
219,
260
Cruchaiid,
S.,
217,
260
Cudkowicz, G., 205, 206, 208, 209, 224,
230, 231,
260, 267
Cudowicz,
G.,
213,
259
Cunniff, R. V.
H.,
96,
109
Cunningham, A., 207, 222, 228, 238,
265,
Cunningham, A.
J.,
205, 268
Cnnningham, B. A., 79, 80, 81, 109, 110,
337, 338, 343,
377
Cunningham, G. J., 309,
324
Cuppavi,
C. ,
321,
330
Curry, J .
L.,
213, 225, 230,
260, 269
Curtis, S., 277, 281
263
263, 306, 310, 324
253, 267, 287, 288, 290, 323, 330
271, 273, 281
Curtoni,
E.
S., 117, 119, 136, 146, 150,
152, 177, 193,
196
D
Daems,
W. T.,
290, 292, 294, 312,
323,
Dagher,
R.
K., 136, 170,
193, 200
Daguillard, F., 209, 215, 216, 225, 233,
Dales,
S. ,
296, 324
Ilalmasso, A. P., 248,
260
Dalrnsso, A. P., 95, 113
Dameshek,
W.,
163,
191
Dammin,
C .
J.,
165,
193, 197
Dandliker,
W . B., 60, 09
Danielli, J. F., 155, 193
Daniels, C.
A.,
96,
109
Daniels, J . R., 305, 306, 327
Danon,
D.,
293, 296,
322
Darzynkiewicz,
Z.,
319, 324
Daugharty, H., 7, 41, 52,
53, 55
Daune, M., 62, 112, 351, 378
Dausset, J., 117, 119,
128,
135, 150, 152,
180, 183,
193, 194, 198, 200
David,
G. S. ,
25,
53
David, J.
R.,
189, 193, 316, 324, 330
Davidson, B., 361,
376, 378
Davidson, E. A., 322
Davie,
J. M.,
19,
53, 55,
71, 72, 98,
109,
377
Ilavies, A. J.
S.,
203, 204, 214, 222, 227,
228, 229, 233, 237, 238, 251, 252,
260, 262,
271, 273,
280, 281,
285,
324
324
245,
246,260,267
Davies, A .
M.,
179,
192,
207,
259
Davies, D.
A.
L., 118, 119, 120, 125, 126,
127, 128, 130, 134, 146, 147, 148,
150, 152, 156, 180, 182, 186, 193,
194, 198, 199, 200
Davies, D. R., 356, 377, 381
Davies,
P.,
305,
324
Davies, W. C., 156, 194
Davson,
H.,
155,
193
Dawson, G., 73, 109
Dayhoff,
M. O., 109
DeBoutaud, F., 103, 104, 109
Decreusefond, C., 206,
259
de Duve, C., 283, 284, 292, 293, 294,
Ilefendi, V., 212, 220, 269
299, 305, 307,
322, 324
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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388 AUTHOR
INDEX
Degani,
D.,
180, 200
Degiovanni,
G.,
136, 195
Dekaris, D., 174,
194
de Koning, J., 256, 260
de la Chapelle,
A.,
179, 195, 316, 325
Delaney, R., 338, 378
Del Guercio,
P.,
97, 109
De Lorenzo, F . , 344, 377
Demopoulos,
R., 168,
199
Den Dulk, M . D., 210, 269
de Petris,
S . ,
290, 324
Dersjant, H., 258
Desai,
I.
D., 305, 324
de Saussure,
V .
A.,
60,
109
Deutsch, H.
F.,
59, 61, 62, 63, 65, 73, 74,
75, 76, 77, 78, 82, 85, 86, 93, 109,
110, 112, 115, 349, 351, 377, 381
Deutsch, L., 167, 194
Deverill, J., 93, 98, 99,
111
Di Cassano, D. L., 215, 259
Dicke, K. A., 247, 248, 256, 260
Diener, E., 206, 225, 228, 252, 258, 273,
Dietrich,
F. M.,
276,
280
Di George, A.
M.,
272, 278, 280
Dingle,
J .
T., 284, 309, 310, 324, 330
Dintzis,
H .
M., 356,
380
Dische, Z., 140, 194
Dixon,, F.
J . ,
38, 56, 171, 194, 202, 203,
228, 251, 260, 269, 274, 275, 276,
277, 279, 280, 285, 288, 311, 324,
327
279
Dobson, E.
L.,
288, 328
Dolder,
F.,
340, 380
Dolon,
M.
F.,
235,
264
Donch,
J . J.,
363, 377
Doolittle, R. F., 9, 50, 51, 53, 56, 66, 79,
Dooren,
L. J.,
256, 260
Doria, G., 209, 210, 232, 258, 260
Dorling,
J. ,
309, 324
Dorner, M . M . , 5, 22, 53, 56
Dorrington, K. J. , 65, 78, 84, 87,
110,
336, 337, 338, 339, 340, 341, 353,
358, 359, 360, 363, 364, 365, 371,
373, 374, 377, 379, 380, 381
Doty,
P.,
218, 262, 287, 304, 325, 357,
362, 378, 380, 381
Douglas, S . D., 220, 262, 290, 317, 323,
324
80, 83, 86, 110, 113
Douglas, P., 162, 199
Dourmashkin,
R.
R., 67, 68, 69, 70, 75,
Dow, D., 321, 329
Dowden, S. J., 231, 260
Draper, L. R., 229, 268
Dray,
S.,
6, 56, 100, 101, 109, 114, 140,
149, 196, 198, 218, 254, 258, 264,
271, 275, 279, 287, 304, 322
Dresser, D.
W.,
162, 171, 194, 202, 205,
228, 251, 254, 260, 268, 286, 287,
324
95, 102,
110,111, 114
Dreyer,
W.
J., 6, 50, 54
Dubert,
J.
M . ,
276,
279, 280
Dubiski, S., 6, 53
Dukor, P., 289, 314, 324
Dumonde, D.
C. ,
207, 261, 309, 316,
324, 327
Dutton,
R.
W., 202, 205, 207, 214, 224,
228, 229, 230,
231,
234, 237, 259,
261, 265, 266, 271, 277, 278, 280,
281,282,
316,324
E
Eady, J. D., 207,261
Easley, C.
W., 76, 77, 78, 80,
114, 380
East, J., 225, 262
Ebert,
R.
H., 311, 313, 325
Edelhoch, H., 66, 113, 114, 115, 344,
351, 379, 381
Edelman, G. M., 1, 2, 10, 11, 53, 79, 80,
81, 99, 102, 103, 104, 109,
110,
113,
333, 337, 338, 341, 342, 343, 345,
346, 349, 350, 351, 371, 372,
377,
379, 380, 381
Edelstein,
S.
J., 337, 380
Edidin, M., 133, 184,194
Edniau, P., 77,
110,
114
Edsall, J. T., 337, 342, 376, 377
Ehrenreich, B. A., 292, 294, 295, 299,
300, 323, 324
Ehrich, W . E., 285, 324
Eichmann, K., 4, 8, 9, 14, 15, 19,
20,
21,
22, 24, 25, 26, 27, 28, 32, 33, 36, 38,
49, 51, 52, 53,
54
Eichwald, E.
J.,
163, 164, 194
Eijsvoogel,
V . P.,
179, 194
Eisen,
H.
N. ,
5, 9, 44, 45, 53, 54, 89, 92,
116, 377
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR
INDEX
389
Eisenberg, H., 337, 376, 380
Eldredge,
J.,
255, 263
Elkins,
W.
L., 194
Ellem,
K. A.
O.,
183,
185,
194
Elliott, E. V., 204, 222, 233, 260, 271,
Ellis, S . T., 271, 280
Ellman,
C.
L., 82, 110
Elson, J., 212, 263
Elves,
M .
W., 179,
194,
317,
323
Engelfriet, C. P., 179, 183, 194
Ephrussi, B.,
118, 199
Epsniark, A., 182, 1.94
Epstein,
H.
F., 66,
116,
346,
381
Epstein, L. B., 136, 137, 178, 179, 196
Epstein, W., 349, 381
Emback, S. , 33, 53, 56
Ernisse, J. G., 117, 186, 200
Eron, L., 58, 115
Essner,
E.,
291,
324, 328
Evans,
D .
G., 291, 324
Evans, E. P., 208, 234, 235,
261, 264
Everett,
N .
B., 208, 261, 266
273, 280
F
Facon, M., 5, 55
Faegraeus, A., 182,
194
Fahey,
J.
L., 2, 54, 59, 100, 101, 108,
110, 119, 131, 132,
133,
135, 147,
148, 150, 151, 152, 153, 155, 197,
201, 225,
261,
340,
377, 380
Fahlberg,
W.,
209, 225, 230, 231, 268
Fanger, H., 316, 324
Farber,
M.
B.,
203,
262
Farnes,
P.,
316, 324
Farquhar, M. G., 306, 307, 330
Fasman, G. D., 361, 376, 378
Fedorko, M . E., 291, 303, 306, 323, 324
Fehr, K., 305, 324
Fcigcn,
C.
A., 183, 194
Feingold, I., 100,
110
Feingold, N., 150, 152,
193
Feinstein,
A.,
4,
54,
68, 69, 70, 74, 75,
82, 83, 102,
108,
110,
215,
260,
347,
352,
355,376, 377
Feinstein,
D.,
339,
377
Feizi, T., 24, 25, 48, 52,
53, 54
Feldman, J . D., 250, 265
Feldman, M., 204, 216, 218, 244, 261,
Fell,
H.
B.,
284, 309, 310,
323, 324
Fellows,
R.
E.,
338, 378
Felton, L. D., 287, 324
Fennel, R. M. ,
311,325
Fenner, F., 170, 193
Fenton, J. W.,
380
Ferber, J. M., 89, 111
Ferrebee, J.
W.,
235, 256,
259, 268
Festenstein,
H.,
179, 194
Fichtelius,
A.,
161,
194
Field, E. D., 248, 261
Filitti-Wumiser,
S.,
61,
63, 75,
78,
97, 98,
110,111,377
Filkins, J.
P.,
305,
325
Filler,
R.
M., 272, 278, 279
Finch,
C.
A , , 256,
268
Finegold, I., 207, 261
Fink,
C.
J . , 314,
327
Finkelstein, M.
S.,
202, 269
Finn,
J.,
175,
191
Finstad, J., 272, 280
Fisher,
D.
B.,
321,
,325
Fisher, W. D., 59, 62, 63, 71, 110
Fishman, M., 202, 218, 222, 258, 261,
266, 271, 275, 279, 280, 285, 287,
296, 304, 322, 325, 329
Fitch, F. W., 271, 273, 281, 282, 288,
289, 326
Fitzgerald, M . G . , 219, 267
Fitzgerald,
P.
H., 316, 329
Flanagan, J . F., 118, 122, 123, 182, 197
Flax, M . H., 183, 194
Fleischman,
J. B.,
1,
2,
10, 14, 19,
54,
Flexner, S., 168, 194
Floersheim, G . L4.,173, 194, 256, 261
Florenti,
I.,
127, 153, 195
Florsdoif, E. W., 136, 193
Forbes, I.
J.,
253,
261
Forbes, J.
G.,
185, 194
Ford,
C.
E., 208, 209, 226, 234, 235, 255,
257, 258, 261, 264
Ford, W.
L.,
212, 218, 220, 234, 237,
259, 261, 286, 325
Foschi, G. V., 146,
197
Foster, J. F., 342, 377
Foster, R., 205,
261, 264
Fougereau, M . , 371, 377
264, 267,
271, 275,
280,
286,
325
363, 377
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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390 AUTHOR
INDEX
Fowler, R., 279,
282
Franco, D., 173, 198
Franek, F., 372,
377
Frangione, B., 2, 3, 54, 74, 80, 83, 87,
Frank, M. M., 74, 92, 96,
110,
288, 289,
Franken-Paris, M., 146, 196
Franklin,
E.
C., 2, 3, 54, 61, 74, 75, 80,
88, 90, 91, 98, 100, 109,
110,
114,
88, 98,
110, 113, 114, 377
290,
326
339,
377
Franks, D., 206, 260
Franzl, R.
E.,
288, 293, 298, 299, 303,
Fred, S . S ., 205,
261
Freedman, M. H . , 367, 368, 369, 377
Freeman,
T.,
101,
109
Frei, P.
C.,
217, 240, 254, 260, 261, 286,
French, V. J., 289, 325, 330
Frenzl,
B.,
117,
199
Friedman, E., 96,
115
Friedman,
E.
A,, 159,
197
Friedman,
H.,
205, 219, 251,
261, 267
Friedman,
H . P.,
287, 304,
325
Friend, D. S., 293, 296, 300, 325
Fronstein, M . H., 277, 280
Fnchs, S., 368, 369, 377
Fudenberg,
H .
H. , 94, 95, 96, 97, 98,
101,
111,
112, 115, 220, 262, 381
Fullmer, H . M., 305, 306, 327
325
325
G
Gabrielsen,
A.
E.,
202, 226,
260, 261,
Gaither, T., 96,
110
Gall,
W. E.,
1, 2,
53,
79,
80,
81, 99, 110,
333, 337, 338, 343, 349, 350, 351,
377, 380
272,
280
Gallagher, J. S., 103,
110
Gallagher, R., 183,
200
Gallily,
L.,
244,
261
Gallily,
R.,
218, 261, 271, 275, 280, 286,
Gally, J. A,, 341, 342, 371, 372,
377
Gangal,
J .
G.,
118, 194
Gaon, J.
A , ,
60,
96, 113
Garcia, G., 240, 254, 259
325
Garvey,
J. S.,
275, 280, 285, 288, 303,
Gatti,
R.
A.,
226, 2.55, 256, 257 ,
261, 264
Gel],
P .
G. H., 6, 7, 11, 53, 54, 99, 112,
179, 198, 199, 215, 267, 306, 316,
323, 329
Gelzer,
J.,
54
Gengozian,
N.,
205, 210, 226, 260, 261
Gentou,
C. ,
61, 97, 98, 110
German, G., 94, 109
Gershon, H., 244, 261
Gershon, R. K., 222, 228, 252, 262, 271,
Gerv,
I.,
179,
192
Gervais, A. G., 156, 194
Gery, I., 207, 252, 259, 268
Gesner, B. M., 209, 235, 262, 269
Gianetto, R., 283, 305, 324
Gibbs, J. E., 248, 261
Gibson, T., 194
Gill,
F.
A., 3001, 325
Gill,
T.
J.,
111,
287, 323, 362, 378
Githens, J. H., 256, 262
Gitlin, D., 101,
110
Giusti, G. V., 226, 260
Givas, J., 40,
54
Givol, D., 216, 267, 344, 369, 373, 377,
Gleich, G. J., 75, 88, 109, 110, 340, 378
Glenchur,
H.,
73,
1 1 1
Glenny, A. T., 171, 194
GliSin,
V. R.,
218, 262, 287, 304, 325
Globerson, A., 216, 242, 243, 261, 262,
267, 268, 269
Godal,
H .
C.,
98,
111
Coebel, W. F., 32, 53
Gotze, O., 315, 326
Gofman, L., 210, 263
Goldberg, B., 183, 185, 194, 195
Goldberg, R. F., 369, 379
Goldschmidt,
P.
R., 305, 315, 330
Goldschneider, I., 235, 249,
262
Goldstein,
A. L.,
226,
262, 263
Goldstein, D .
J.,
356, 357,
377, 380
Golub,
E .
S.,
313,
325
Gonatas, N . K . , 317,
329
Good,
R.
A., 106, 111, 171, 172, 195, 196,
197, 199, 202, 226, 229, 247, 248,
249, 251, 255, 256, 257, 260, 261,
323
280
378, 380
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR
INDEX 391
262, 264, 265, 268, 270, 272, 279,
280, 281, 282, 313, 326, 380
Goodman,
H.
S.,
185,
194
Goodman,
J. W.,
210, 211, 219, 220, 235,
260, 262, 267, 363, 377
Gordon,
A.
S., 155, 200
Gordon,
G.
B.,
286, 298, 325
Gordon, J.,
171, 195,
207, 221,
262, 325
Gordon,
S.,
83, 109, 111, 371, 377
Gorer, P. A,, 156, 162, 168, 176, 181, 182,
183,194, 197
Gorguraki,
V.,
351,
378
Gorini, G., 6, 53
Gotschlich,
E.
C.,
46, 48, 49,
54
Gottlieb, A. A., 218, 222, 262, 287, 302,
304,
322, 325
Gottlieb,
C.
F., 205, 261
Gottlieb, P. D., 79,
80, 81,
110,337, 338,
343, 377
Gough, J., 317,
323
Gould, H. J., 362, 378
Goulian, D., 288, 325
Gowans, J. L., 173, 197, 209, 211, 212,
218, 223, 231, 234, 237, 247, 250,
251, 261, 262, 263, 264, 269, 271,
280,
286,
325, 329
Gowland, G., 161, 171, 172, 192, 194
Grab,
B., 60
14
Grabar, P., 119, 136, 195
Graetzer,
M.
A, , 272, 279
Graff,
R.
J., 117, 146, 148, 159, 170, 182,
Graham, R. C., Jr., 292, 295, 302, 308,
Granger, G.
A.,
221,
262, 269,
316,
325
Grant,
L.
E., 311, 313,
325
Granthana, W.
G.,
212, 259
Grasbeck, R., 179, 195, 316, 324
Grate, H. E., 208, 231, 262
Gray, J., 208, 23S, 264
Gray,
J .
G., 261
Gray, W.
R.,
6, 50,
54
Greaves, M . F., 105, 111, 215, 246, 262
Green, D. E., 136, 199
Green,
H.,
183, 185, 194, 195
Green, I., 276, 278, 280
Green, N. M., 67, 68, 70, 1 1 1 , 347, 348,
Greenblatt,
J. ,
17, 33,
53,
5 4
Greenfield, N., 361, 378
195
323, 325, 327
378, 381
Greengard, P., 321, 328
Greenwood,
F. C.,
74,
110
Grey,
H.
M.,
2, 3, 7, 54,
68,
79, 83, 102,
108, 111, 203, 262, 339, 349, 371,
372, 376, 378,
380
Griffith, R.,
340, 378
Groff,J. L., 89, 111
Crogg, E., 301,
324
Grossberg, A. L., 64, 3, 91, 92, 114, 355,
Crossman,
J.,
314, 321, 326
Groves,
D. L.,
271,
281
Grubb, R., 6, 54
Gruchaud,
S.,
254,
261
Gruneberg, H., 165, 195
Grunnet,
I.,
209,
259
Gruskin,
R.
H., 179, 195
Gudat,
F. G.,
100,
1 1 1
Gunderson, C. H., 271, 282
Gumer,
B.
W., 217,
263
Guttman, R. D., 172, 19.5
379
H
Haager, O., 343, 349, 351, 380
Haas, E., 136, 195
Habeeb,
A . F.
A., 78,
111
Haber, E., 2, 10, 12, 29, 30, 32, 37, 38,
40, 48, 54,
56,
358, 359, 360, 363,
367, 372, 376, 378
Habermeyer, J.
G.,
255, 265
Habich, H., 99, 111
Hade, E.
P.
K.,
337,
378
Haenen-Severyns,
A.
M., 136, 195
Haenen-Severyns, H.
A.,
146, 196
Haimovich, J., 60, 89, 94,
111,
114,
216,
Halasz, N.
A.,
172, 173, 195, 199
Hall, J. G., 237, 262
Hallenbeck,
G .
A., 256, 265
Halle-Panenko, O., 127, 153, 195
Halpern,
B.,
317,
325
Hamaguchi,
K.,
359, 378
Hamerton, J. L., 208, 255,
261
Han, J.
H.,
290, 325
Han,
S. S.,
221,
269,
290,
325
Hanan, C., 183, 197
Hanks, J.
H.,
183,
195
Hanna, M. G., Jr., 283, 326
Hiinnoiin,
C.,
290,
326
Hansrn,
S .
S., 95, 112
267
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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392
AUTHOR INDEX
Hanson, L. A,, 378
Harboe, M., 85, 93, 95, 98, 99,
111
Hardy,
j.
D.,
179,
198
Hargrave, D.
C.,
173, 198
Harinovich,
J.,
32, 56
Harris, G., 221, 253, 262
Harris,
J.,
124, 167, 195
Harris, J. E., 262
Harris, P.
F.,
206, 208, 209, 262
Harris, R. S., 206, 208, 262
Harris, S., 100, 111, 203, 262
Harris,
T. N.,
100, 111,
Hartmann,
L.,
61, 63, 75,
Harvard, R . J., 171, 195
Hasek, M., 202,
262
Haskill, J.
S.,
224, 228,
234,
262, 265
Haskova. V.. 145. 195
203, 262, 285, 324
111,
377
124, 167, 195,
78, 97, 98,
11
0,
229, 230, 233,
, r I
Hathaway,
W. E.,
256, 262
Hauge, M., 150, 152, 196
Haughton, G., 119, 136, 156,
183,
191,
Haupt, H., 63, 111
Haurowitz,
F.,
303, 326
Haurowitz, H., 276,
280
Hauschka,
T.
S. , 118, 199
Hauser, K.
E.,
290,
326
Haxby, J.
A.,
3 1 5 , 3 2 6
Hayes,
C .
R., 170,
200
Hazen,
R.,
59, 75, 83, 111
Heersche,
J. N.
M.,
194
Hege, J. S., 205, 262
Hegner,
D.,
309,
326
Heide, K., 63,
11
1
Heidelberger,
M.,
29, 54, 58,
111,
378
Heilman, D. H., 244, 264
Heiniburger, N., 63, 11
1
Heinzler,
F.,
62, 112, 351, 378
Heise, E. R., 221, 269, 301, 326
Heller,
P.,
87, 92,
101, 109, 116
Hellman, S., 208, 231,
262
Henimingsen, H., 224, 237, 239,241, 242,
243, 246,
266, 268,
271, 272, 273,
278, 282
195
Hendree, E. D., 136, 198
Henney, C .
S.,
275, 280
Henry, C., 106, 111
Henson, P. M., 97, 111,
311,
326
Herbst, M., 343, 349, 351,
380
Heremans, J. F., 59, 64, 76, 109, 114,
Herion, J.
C.,
306, 326
Hersh,
E.
M., 262
Hertenstein, C., 65, 11
6
Herzenberg, Leonard
A, ,
6, 54, 146, 195
Herzenberg, Leonore A., 146, 195
Hesch, J.
A,,
256,
258
Heslop, B.
F.,
169, 195
Hess, M .
W.,
202,
262
High,
G.
J., 305, 306, 330
Hijnians, W., 101,
116,
210,
269
Hildegard, H.
R.,
270
Hildemann,
W.
H.,
115, 117,
165, 169,
Hilgard, H., 249,
268
Hilgert, I., 121, 171, 195, 196
Hill, J. H., 305, 315,
326, 330
Hill, R. L., 338, 378
Hilschmann, N., 25, 54
Hirata,
A.
A., 185, 195
Hirose, F., 172, 195
Hirsch, J.
G.,
291, 306, 307, 310,
322,
Hirschhorn, K., 179, 195, 290, 317,
318,
Hirschhorn,
R.,
290, 314, 316, 317, 318,
Hirsh, E. M., 286, 328
Hirvonen, T., 101, 115
Hoak, J. L., 136, 193
Hobson, D., 210, 262
Hochwald,
G.
M., 248, 260
Hodgson,
G.
S.,
235,
262
Hoecker, G., 117, 168, 199
Hoelzl-Wallach, D. F., 156, 198
Hoffman,
P. F.,
317, 324
Hogebroom, G.
H.,
136, 195
Hogg, N. M., 81, 114
Holborow, E. J., 149, 195
Holm,
G.,
189,
195, 198
Holman, H. R., 91, 110
Holnies, B., 313,
326
Holmes, E. C., 119, 136, 144, 196
Holnb, M.,
119,262,
290,
326
Hong, R., 226, 255, 256, 2S7, 261, 264,
370, 372, 378, 379, 380
Hood, L., 4, 6, 8, 9, 19, 20, 21, 22, 24,
27, 28, 32, 50, 51,
53, 54,
77, 80,
81, 99, 111, 112
339,
378,
379, 381
181, 195
323, 324, 329
319 , 321 , 323 ,
326
319, 320, 321,
323, 326, 330
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX 393
Hopkins, B. E., 171, 194
Hopper, J. E., 7, 41, 52,
53, 55
Horibata,
K.,
229,
258
Horinchi,
Y . , 108
Hornbrook, M. M., 378
Houser,
R.
E., 219,
262
Howard, J. G . , 212, 220, 259, 262, 263,
271,
280,
287,
326
Howell, S. L., 314, 327
Howson,
W. T.,
207,261
Hoyer, L.
W.,
64, 95, 1 1 1
Hraba,
T.,
202, 205,
262
Hrubeskova, M., 145, 195
Hu,
C. C.,
174,
193
Huber, H., 96, 97, 111, 220, 262
Hudson,
G . ,
208, 209,
262, 270
Hughes, 1).
E.,
136, 195
Hughes,
W .
L., 208, 209,
260
Huisman,
T.
H. J., 59, 113
Hulliger, L., 301,
329
Humphrey, J. H., 6, 38, 53, 54, 70, 74,
92, 95, 96, 106, 110, 1 1 1 , 225,
262,
287, 288, 289, 290, 326, 327
Humphrey, R .
L.,
356, 357,
377
Humphreys, T., 118, 195
Hunt,
W. L., 6 , -56
Hunter,
A.,
88, 1 1 1
Hunter, R. L . , 288, 289,
326
Hurley, J.
V.,
315, 326
Hii\bantl, E .
M.,
179,
197
Hutas, Z., 210, 263
Hutchison,
M., 182,
193
Hntt, M . S.
R.,
288, 329
Hystop, N. E., 95, 1 1 1
I
Ihler, G., 58,
115
Ikeila, K., 359, 378
Ikenaka,
T., 112,
351,
378
Imahori, K., 362, 378
Iinamura, T.,
204,
263
Ingraham, J.
S. ,
205, 263
Ingrain,
P.,
345,
378
Inman,
F.
P.,
59,
61,
74, 75, 83, 99,
111,
113.
115
Inoue, K., 96, 1 1 1
Ippen, K.,
Fj8,
115
Irvine, R. A., 73,
113
Isakovic, K., 252,
263
Iscaki, S . , 363, 379
Iseri,
0.A . ,
307,
327
Ishizaka,
K.,
94, 95,
11
I 275, 280, 309,
Ishizaka, T., 94, 95, 111, 309, 326, 351,
Isliker, H., 93, 112
Israels,
M . C.
G., 179,
194
Ihvin,
W.
s.,
183, 194
Ivang, P., 117,
193
Ivany, D., 117, 193
Ivanyi,
J.,
215,
267
326,
351,378
378
J
Jackson, J. F., 179, 198
Jacobson, E. B.,
248,
260
Jacobson,
L. O., 255, 263
Jacot-Guillarmod, H., 93,
112,
275,
280
Jacques,
P.
J., 285, 290, 326
Jafk'e, P. W., 117,
195
Jahnke,
V. K.,
62,
112,
351,
378
Jamison, G.
A.,
147,
198
Janeway, C.
A.,
Jr., 171,
195,
216,
264,
Jankovic,
B. D.,
250,
263,
272,
280
Janoff,
A , ,
305, 306, 309, 311, 313, 326,
Jaroslow,
B. N., 244, 268, 289, 327
Jaton,
J. C.,
12, 29, 56, 89, 112, 369, 378
Jeannet, M., 256,
263
Jenkin,
C. R.,
221,
264,
301,
327
Jenkins, G. C., 173,
200
Jensen,
C.,
186,
195
Jensen, E., 167,
199
Jensen,
K.
G.,
186,
195
Jerne, N.
K.,
106, 111, 118, 189, 190,
Jerrard, H.
G.,
345,
378
Jirgensons,
B.,
65, 71,
112,
343, 351, 358,
359, 362, 378, 380
Jobe, A. ,
44,
56
Jobling, J. W., 168, 194
Johansson,
B.
G.,
376
Johansson,
S . G .
O., 101, 112, 340, 341,
Johnke, K., 65, 116
Johnscm, D. O., 289,
328
Johnson,
A .
C. , lG2, 197, 290, 325
Johnson,
B. W.,
117, 189,
197
Johnson, L.
W.,
117, 193
Johnson, P., 344,
376
272, 278,
279, 280,
287,
326
327,329
195, 205,
238,
246,
263,
277,
280
376, 378
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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394
AUTHOR INDEX
Johnson, V., 277, 281
Jolles, P., 127, 153, 195
Jones,
H. B.,
288,
328
Jones, V. E., 277, 281
Joo,
P.,
255, 256, 257, 258
Jureziz, R., 41, 55, 356, 379
K
Kabat, E.
A,, 5,
10, 11 , 16, 22, 48, 53, 54,
55, 56, 58, 62, 63, 64, 89, 112, 113,
378
Kahan, B. D., 119, 126, 136, 137, 138,
141, 142, 144, 145, 146, 149, 154,
155,
159, 161, 162, 163, 165, 166,
174, 177, 178, 179, 180, 188, 191,
195, 196, 198, 200
Kalfayan, B., 183, 196
Kaliss, N., 168, 194, 196
Kaltenbach, J.
P.,
183, 196
Kaltenbach, M. H., 183, 196
K a m a j t o v i , V., 290,326
Kamentsky, L. A., 183, 197
Kaminski, M., 304, 327
Kamrin,
B.
B.,
173,
196
Kandutsch,
A.
A., 118, 120, 121, 124,
146, 147, 148, 159, 169, 170, 173,
182, 195, 196
Kane, J. A., 48, 54
Kaplan,
A. P.,
64, 65, 77, 80, 81, 108,
Kaplan, J. G., 317, 321, 326, 329
Kaplan, M . E., 89, 112, 288, 323
Karakawa,
W. W.,
12, 17, 18, 48, 54, 55
Karlin, L., 64, 0, 93, 113, 355, 356, 379
Karlsbad,
G.,
290,
324
Karlsson, F. A,, 349, 378
Karnovsky, M. J., 251, 259, 313, 322
Karnovsky, M.
L.,
313, 322
Karthigasu, K., 301, 327
Kauffnian, G., 287, 324
Karush,
F.,
50,
56,
336, 363, 380, 381
Kasakura, S., 207, 245, 263
Katz, M., 30, 56
Kawasaki, M., 108
Keiser,
G.,
208, 263
Keller, H. U., 106, 111 , 305, 315, 322
Kellermeyer,
R. W. ,
302, 308, 325, 327
Kelly,
L.
S., 208, 267
Kelly,
W.
D., 171,
196
Kelus, A. S.,
7,
11, 54, 99, 101, 112, 114
112
Kenh, J.
E.,
336, 380
Kennedy, J. C., 205, 214,
263
Kenny, K., 94,
114
Kent,
G.,
317,
327
Kerby, C.
C.,
235, 264
Keuning, F. J., 210, 268
Kidd, J. G., 183, 196
Kierszenbaum, F., 60, 109
Kiger, N., 127, 153, 195
Killander, D., 319, 320, 327, 329
Killander, J., 349, 380
Kimura, J., 50 , 56
Kind,
P.,
205, 229, 263
Kindt,
T.
J., 6, 8, 9, 19 , 24, 25, 26, 36,
King, D. W., 207,
267
Kinneart,
P.,
136, 170, 193, 200
Kinoshita, Y., 108
Kinsky, R. G., 212, 220, 262, 263
Kinsky, S. C., 315, 326
Kirchmyer, R., 205, 261
Kishimoto, T., 59, 74, 84, 86, 87, 112,
Kissmeyer-Nielsen,
F.,
150, 152,
196
Klapper,
D. G.,
103, 104, 112
Klein, F., 88, 112
Klein,
G.,
143, 144, 171, 185, 196
Klein, J., 171, 196
Kleinsmith, L. J., 319, 327
Klinman, N. R., 50, 56, 336, 369, 378,
Kluge, J. P., 301, 328
Knight, E. J., 234, 262
Knopp, J. A., 66, 112
Knyszynski,
A,,
226, 227,
259
Kijhler, H., 80, 81, 112
K6lsch, E., 292, 294, 295, 300, 327
Kohler, H., 64, 78, 79, 80, 81, 83, 116
Kolb,
W. P.,
316, 325
Koller, P. C., 204, 214, 222,
228, 233,
237, 238, 260,
271,280
Kolodny, E . H., 140, 196
Kontiainen, S.,
89, 105,
112
Kopecky, K. E., 301, 328
Koponen,
T.,
89, 105, 112
Korach,
S.,
88,
114
Kornfeld, L., 219, 263
Korngold, L., 75, 112
Koshland,
M. E., 6,
S4, 140,
196
Kostiainen, E., 89, 105, 112
51, 54
114
380
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX
395
Kosunen, T. U., 251, 263
Konrilsky,
R.,
317,
325
Kovacs,
A .
M. , 62,
112,
351,
378
Kozuru,
M.,
76, 77, 78, 80,
114, 380
Krakauer, K., 305, 324
Kratky,
O.,
343, 349, 351,
378, 380
Krause, R .
M.,
4, 8, 9, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 24, 25, 27,
28, 32, 36, 37, 38, 48,
49,
51, 52,
53, 54, 55
Krause, S., 345, 378
Kren, V., 117, 199
Kretschmer,
R.,
256, 263
Kretshnier,
R.,
278,
280
Krinsky,
R.,
168, 169, 200
Kdstiansen,
J.,
33, 55
Kritznian,
J.,
43, 55
Kruse, H., 288, 327
Kugler,
J.
H., 206, 208, 209, 262
Kulczycki, A, , 359, 360, 376
Kumate,
J.,
101, 110
Kunkel, H. G., 6, 7, 10, 11, 43, 53, 54,
55, 56, 59, 61, 65, 71, 88, 90, 91, 93,
100.
110.
112, 113, 114,
115,
116,
LaVia,
M . F.,
271, 282
Law, C , . R.
,,
267
Law,
L. W., 223, P25, 220, 229, 256,
261,
263, 268, 269
Lawrence, H .
S.,
119, 135, 160, 163, 191,
198, 207, 269, 316, 327, 330
Lawrence,
J . S. ,
242, 260
Lay, W. H . , 97, 112
Lazarus,
G .
S., 305, 306, 327
Leake, E. S., 291, 301, 326, 327
Leblond,
C .
P., 235,
267
Lebovitz, H . E., 338, 378
Lederberg,
J.,
183, 198
Lederberg,
S.
B.,
184,
196
Lederberg, V., 184, 196
Leduc, E. H. , 288, 323
Legge, D.
G.,
234, 262
Legrand, L., 150, 152, 193
Lehniann, R. P., 301, 328
Lehrfield, A. W., 164, 200
Lehrfield, J., 200
Lejeune,
G.,
136, 146, 195, 196
Lengerova, A,, 202,262
Lennox,
E .
S., 5,
5.5,
229,
25 ,
333, 338,
339;
349;
378,' 380
Kuo, J . F., 321, 328
Kyle,
R.
A., 256, 265
L
Lachmann, P.
J.,
206,
Lack,
C.
H., 305, 322
324, 327
379
Leon, M., 46, 55, 56
Leonard,
hl. R.,
291, 329
Lepinay,
A.,
317, 325
Lepow, I.
H . ,
95, 113, 305, 315, 330
263, 310, 315, Leskowitz, S., 202, 263, 277, 281, 287,
327
Leslie,
G.
A., 102, 103, 109, 112
Lackland, H., 4, 8, 9, 19, 20, 21, 22, 24,
Lacy,
P.
E.,
314,
327
Lagunoff, D., 291, 307, 327
Lnnim,
M.
E., 63, 75, 76, 77, 78,
112,
Lamp], H., 276, 281
Lance, E. M. , 173, 196
Lancefield,
R.
C., 13, 14, 37, 55
Landschuetz,
C.,
183,
196
Landsteiner, K., 276,
280, 281
Landy,
M.,
277, 278,
281,
316,
327
Lang,
P.
G.,
277,
281,
287, 289, 292,
Larsen, A . B., 301, 328
Larsen,
A.
E., 38, 56
Lasky, L. J . , 285, 323
27, 28, 32, 51, 53, 53
335,
336, 337, 372, 379, 380
293, 295, 299, 300, 322, 328
Leuchars, E., 204, 214, 222, 228, 233,
237, 238, 251, 252, 260, 262, 271,
273,
280
Leventhal, B. G., 288, 328
Levine,
B.,
175, 196, 277, 281, 307, 308,
Levinson, W.
E.,
96, 112
Levis, W . R., 286, 327
Levison,
S.
A., GO 109
Levy, D.
A.,
314, 327
Lewis, P.
A.,
175, 191, 200
Liacopoulos, P., 173, 191
Liacopolous-Briot,
M.,
317,
325
Lichtenstein, L.
M.,
314, 327
Lichter,
E. A.,
99,
132
Liden, S., 235, 251,
263
Likhite, V., 221,
263
313, 327
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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396 AUTHOR INDEX
Limpert, S., 302, 325
Lind,
I., 220, 266,
292, 293,
295, 329
Lind, P. E., 279,
282
Linder, O., 171, 197
Lindqvist,
K.,
91, 112, 355, 379
Lindsley, D. L., 255, 263
Ling, N .
R.,
179, 197
Linna,
T.
J., 235, 251, 263
Linscott, W. D., 95, 96, 97, 98,
111,
112,
Litt, M., 307, 327
Littau, V. C., 320, 322
Little, C. C., 117, 162, 189, 197
Little,
J. R.,
42, 44,
54,
55,
377
Litwin, S. P., 6,
55
Lin, T.
Y.,
46, 48, 49, 54
Llacer, V., 321, 330
Lochte, Ii. L., 256, 268
Loeb, L., 117, 157, 175, 189, 197
Loegering, D.
A.,
75, 110
Loewi, G., 149, 195, 220, 263, 309,324
Lomhos,
O.,
210, 263
Longmire, W. P., 176, 183, 200
Loomis,
D.,
175,
197
Lorenz, E., 255, 263
Lospalluto, J., 88, 112, 305, 324
Loutit, J . F., 208, 212, 234, 255, 256,
257,
258, 261, 263
Lovett, C . A., 312, 316, 327
Lowenstein,
L.,
179, 191,
192,
207, 245,
Lowey, S., 65, 115, 358, 359, 380
Lubaroff, D. M., 207, 249, 250, 263
Ludke,
H.,
210, 263
Lukes,
R.
J., 316,
329
Lnmnius, Z., 254, 263, 289, 328
Lurie, M., 290, 323
Lustgraaf,
E.
C.,
163,
164,
194
Luxemberg, K. I., 94, 109
Lycette, R .
R.,
179,
197,
316,
329
Lyman,
S.
A,, 162, 194
Lyon,
M .
F., 255,259
Lyons, W . B., 183, 196
114, 220, 262, 278, 281
258, 263
M
McCarthy, J., 43, 55
McCarty, M., 14, 15, 16, 17, 37,
55
McClelland, J. D., 176, 183, 186, 200
McCluskey, J. W., 250, 251, 263
McCluskey,
R.
T., 250, 251, 260, 263,
McConahey,
P.
J.,
275, 277,
279, 280,
McConnell, I., 217, 263
McCullagh,
P. J.,
218, 251,
261, 263,
264, 286, 325
McCulloch,
E.
A,, 204, 205, 206, 208,
210, 214, 228, 231, 259, 263, 267,
268, 269, 270
289, 323
288,
327
McCullough, N .
B.,
177, 197
McDevitt,
H.
O., 40,
55,
189, 197, 288,
MacDonald,
A.
B.,
7, 41, 52,
53,
55
McDougall, E. I., 75, 112
McFarland, W., 244,
264
MeGregor, D. D., 173, 197, 223, 231,
Machattie, L., 58, 115
McIntire,
K. R., 68,
70, 115
McIntyre, J., 221, 264
McIvor, K., 221, 269
Mackaness,
G.
B., 202, 221, 264, 285,
McKenna,
J.
M., 252, 258
Mackenzie, M . R., 95, 98, 112
McKhann,
C.
F., 160, 171, 197, 254, 264
McLaughlin, C.
L.,
77, 88, 115, 349, 380
Maclaurin, B. P., 244, 264
McLaverty,
B.,
209, 259
MacLean, L. D., 207, 262
McMaster, P.
D.,
288,
327
Macniorine, D.
R.
L., 306, 309, 312, 323,
McQueen,
J.
D., 292, 295, 300,
328
Madera-Orsini, F., 317, 327
Makela, O., 89, 105, 112, 202, 229, 264,
265, 278, 281, 379
Magar,
M. E., 361, 379
Mage, R., 254, 263, 264
Mahoney, J. F., 256, 258
Maini,
R. N.,
316, 327
Maja, M., 215, 259
Makinodan,
T.,
161, 167, 193, 202, 203,
210, 213, 214, 226, 231, 245, 255,
261, 264, 266, 269, 271, 281
289, 290, 327
235, 247, 249, 251, 254, 262
301,
327
327, 328
Malawista,
S.
E., 314, 315, 327
Malinin, T.
I.,
235, 264
Malomrit,
N., 168,
196
Maloney, M. A., 208, 264
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX 397
Malucci,
L.,
316, 317, 322
Manaker,
R.
A., 207,
261
Mandel,
M .
A,,
203,
26
Mandel,
T.,
106,
108
Mandelkern, L., 335, 380
Mandema, E., 59,
113
Mandy, W. J., 6, 26, 54, 56, 381
Mangalo,
R.,
363,
379
Mann,
D.
L., 119, 131, 132, 133, 135,
147, 148, 150, 151, 152, 153, 155,
197
Mann, L. T., Jr., 165,
193, 197,
287,
323
Mannik, M., 7, 10, 54,
55,
83, 97, 111,
Manning, M., 40, 54
Manson, L. A., 118, 123, 124, 146, 163,
Manstone, A. J., 128,
194
Many,
A.,
252, 264
Marbrook, J., 205, 229,
264
Marchalonis, J. J., 102, 103, 104, 113,
Marchant, R., 204, 222, 233, 260, 271,
Marcuson, E. C., 254, 264
Margaret, J. P., 96, 97,
111
Margaretten, W., 292, 294, 331
Margolies,
M . N.,
29,
56
Margolis, S., 314, 327
Mariani,
T.,
171,
197
Markowski, B., 272, 278, 279
Marks,
E. K.,
255,
263
Marks, J., 206, 260
Marler, E., 335, 337,
379
Marrack,
J.
R.,
103,
114
Marshall, A.
H.
E., 202, 235, 250,
265
Marshall, D. C., 159, 197
Martensson,
L.,
24, 55
Martin, C. M., 177, 197
Martin,
D.,
217,
259
Martin, N. H., 59, 62, 65, 109, 113
Martin,
R.
R., 307, 313,
323
Martin,
W.
J., 285, 327
Martinez,
C.,
171, 172,
196, 197,
199,
Marting,
C.,
171,
195
Massaro, A.
L.,
215, 259
Masson, P. L.,
379
Masurel, M., 118, 123, 148, 193
Mathe, G., 256, 264
113,
371, 372,
378, 379
166, 180, 182, 197, 198
379
273,
280
248, 249, 251, 260, 264, 268, 270
Matheson, D. M., 95,
111
Matsnmato, S., 64, 90, 93, 113, 355, 356,
Matsuyama, M., 235,
264
Matsuyuki, Y., 242, 260
Mattern, P.,
88,
112, 114
Matthew, M., 207, 261
Matthews, 3 W., 356,
381
Mattiuz,
P. L.,
117, 119, 136, 146, 150,
Maurer, P. H., 171, 194, 218, 266, 274,
May, C., 307, 308, 313, 327
Mayer, M. M., 96,
115
Medawar,
P.
B., 118, 119, 123, 136, 145,
146, 147, 157, 158, 159, 160, 162,
163, 164, 165, 169, 170, 172, 173,
174, 176, 178, 181, 182, 192, 194,
195, 196, 197, 251, 259
Mego, J . L., 292, 295, 300, 328
Mekori, T.,
204,
264
Melamed, M.
R.,
183,
197
Melchers,
F.,
5,
55
Mellors,
A.,
96,
115
Mellors, R. C., 43, 55
Melmon, K. L., 305, 309,
328
Mendes, N. F., 118, 122, 123, 182, 197
Merchant, B., 205, 262
Merchant,
D.
J., 118, 194
Mereu, T. R., 256, 263
Merkal, R.
S.,
301, 328
Merkel, F. K., 256, 265
Merler, E., 64, 0, 93, 113, 216, 264, 355,
Merrill,
J.
P.,
159, 183,
197
Merritt, K., 162, 197
Mertens, E., 285, 324
Messina, V. P., 161, 197
Metaxas, M. N., 178, 197
Metaxas-Buchler, N., 178, 197
Metcalf, D., 202, 205, 235, 261, 264
Metchnikofk',
E.,
283, 285, 312,
328
Metzgar, R.
J.,
118, 122, 123, 182,
197
hletzger,
H.,
43,
55,
58, 59, 60, 61, 62,
63, 64, 65, 66, 71, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 90, 94,
99, 100, 105, 107, 108, 110, 112,
113, 115,
351, 353, 355, 363,
379,
381
379
152, 177,
193, 196
276, 277, 280, 281, 304, 329
356, 379
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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398
AUTHOR INDEX
Meuwissen, H. J., 226, 247, 255, 256,
Meyer,
R. K.,
272, 279
Michaelides, M.
C.,
44, 45, 54
Michel, M., 7, 52,
55
Micklem, H.
S.,
208, 209, 226, 234, 235,
Midgely, R., 247, 266
Miescher,
P. A.,
316, 330
Miggaino, V., 117, 150, 152, 193
Migita,
S.,
359, 378
Mihaesco, C., 64, 4, 78, 82, 83, 84, 86,
87, 110,
113, 115,
337,353,377, 379
Mihaesco,
E.,
82, 83, 84, 87, 113
Mihajlovic,
F.,
176, 200
Mikulska, Z.
B.,
156, 181, 182, 194
Milanesi, S., 290, 329
Milgrom, F., 182, 191
Miller, E. J., 12, 17, 18, 19, 48,
53, 55,
83,
113
Miller,
F.,
59, 61, 62, 63, 64, 71, 74, 75,
76, 77, 78, 82, 83, 84, 85, 86, 107,
113,
351, 353, 355, 379
Miller,
J. F.
A.
P.,
173, 197, 202, 203,
207, 210, 214, 222, 223, 226, 227,
228, 229, 233, 2.35, 237, 238, 242,
248,
250,
260, 264, 265, 266, 271,
273, 278, 281, 285, 328
257, 261, 264, 272, 281
257, 258, 261, 264
Miller,
J. J.,
111, 289, 328
Miller,
L.
R., 298, 325
Mills,
J.
A., 179, 198, 207, 265
Mills,
S.
D., 256, 265
Milstein, C., 2, 53, 79, 80, 82, 87, 110,
114, 333, 376
Minick, 0. T., 317, 327
Mirsky, A. E., 317, 319, 320, 322, 327,
329
Mishell, R. I., 205, 214, 224, 229, 230,
234,
237, 261, 265, 266, 271, 277,
278, 280, 281, 282
Mitchell, G. F., 203, 207, 210, 214, 222,
223, 227, 228, 229, 233, 237, 238,
242, 264, 265, 271, 273, 278, 281,
285, 328
Mitchell,
J.,
254, 265, 288, 289, 293, 298,
301, 302,328
Mitchison,
N. A,,
159, 165, 167, 171, 192,
194, 198, 202, 214, 216, 218, 221,
222, 244, 251, 253, 254, 255, 260,
265, 287, 292, 294, 295,
300,
303,
304, 316, 324,327,328
Mitus,
W.
J.,
163,
191
Miyamoto, E., 321, 328
Modabber, F., 185, 199
Moller,
E.,
118,
198,
219, 265
Mdler, G., 105, 106, 116, 156, 198, 202,
Moller,
J.,
118, 198
Moffatt, D. J., 208, 212, 265
Moffitt,
W.,
65, 113, 362, 379
Momoi, H., 378
Monaco,
A.
P., 159, 161, 162, 163, 165,
Montgomery, P.
C.,
36, 358, 360, 379,
Moon, H.
D.,
206,267
Moon, H. G., 293, 296, 300, 325
Moore,
G.
E., 100, 115, 142, 198, 204,
256, 263, 265
Moore, M. A. S., 212, 265
Moore, R. D., 244,267
Moorehead, J . F., 244, 264
Morales,
C.,
101,
110
Morel],
A.
G., 73, 113
Morello, J. A., 299, 303, 325
Moreno,
C., 5,
55, 89, 113
Morita, T., 244, 266, 291, 329
Morley, J., 207, 261
Morris, B., 237, 262
Morris, J.
E.,
59, 61, 74, 83, 113
Morrison, J. H., 208, 265
Morton, D., 119, 136, 144, 196
Morton, J. I., 59, 61, 65, 73, 85, 110, 377
Morton,
R. K.,
124, 198
Morton,
W.
R.
M., 209, 259
Moscona, A. A, , 118,198
Moses, J., 311, 313, 325
Mosier, D. E., 205, 214, 218, 229, 237,
239, 265, 271, 273, 277, 281, 286,
328
269,275,279, 281
172, 173, 198
380
Mosimann, J. E., 71, 78,
113
Moulux,
W.
S., 301, 328
Moura Nuns,
J.
F. B., 317, 322
Mouton,
D.,
206, 259
Movat, H. Z., 306, 309, 312, 316, 323,
327, 328, 330
Mowbray,
J.
F., 173, 198
Moynihan, P., 179, 198
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX
399
Mueller,
G .
C., 321, 325
Miiller-Eherhard, H. J., 59, 61, 91, 94,
95, 96, 97, 109,
110,
111,
113,
116,
220, 262, 312, 315, 326, 328
Mulahasanovic, M., 60, 96, 113
Mumaw,
V.
R., 2 4 4 , 2 6 7
Munn, E.
A.,
68, 69, 70, 102, 110, 315,
Munro,
A.,
217, 263
Munster,
A.
M., 172, 198
Murray, E. S., 60, 96, 113
Murray, J., 161, 198
Murray, R. G., 235, 265
Murphy, J.
B.,
255,265
Mustard, J. F., 312, 328
Mutlu, K. S., 290, 293, 295, 323
Myrvik, Q. N., 291, 301, 324, 326, 327
Myszkowska,
K.,
349, 381
327,
3 5 2 , 3 7 7
N
Najarian, J. S., 250, 256, 265
Nakamura, R. M., 305, 306, 330
Nakano, M., 162, 192
Naor,
D.,
106,
113, 114,
216, 246,
265,
Naspitz, C. K., 207, 244, 265, 266
Nathenson, S. G., 119, 125, 130, 131, 132,
133, 134, 135, 146, 147, 148, 150,
151, 152, 153, 154, 155, 186, 197,
198, 199
Natsuk, W. L., 248, 267
Natvig,
J . B., 6, 55
Nelson, C.
A.,
335, 337, 341, 342, 343,
350, 379
Nelson, D.
S.,
97,
114,
169,
198,
206,
220, 221, 237,
265,
285, 289, 291,
300, 301, 328
Nelson,
E. L.,
301, 322
Nelson, R. A., 96, 97,
111,
114
Neselof, C., 273, 278, 281
Neta,
R.,
309, 327
Nettesheim, P., 244,
266,
291,
329
Neumann, H., 369, 379
Newcomb,
R.
W.,
379
Newlin, C., 219, 267
Nezlin, R.
S.,
345, 371, 372,
377
Niall,
H.
D.,
77,
114
Nind,
A. A,
P.,
220,
263
Nisbet, N. W., 247, 265
Nishioka,
K.,
96,
114
268
Nisonoff,
A.,
7, 26, 29, 41, 52, 53,
55,
56, 140,
149,
198, 356, 370, 372,
378, 379, 380, 381
Norlken, M.
E.,
335, 341, 342, 343, 350,
367, 379
Noltenius,
H.,
277,
281,
290,
328
Nordin,
A. A.,
205, 238, 263
Nordman, C.
T.,
179, 195, 316, 324
Normansell,
D.
E., 379
Norris, H., 183, 198
North, R. J., 221, 237,
265,
291, 328
Nossal,
G. J . V., 100, 114,
183,
198,
202,
207, 222, 228, 229, 231, 238, 254,
265,
271, 273,
281,
288, 289, 290,
293, 298, 301, 302, 322, 327, 379
Notani,
G.,
46, 53
Notkins,
A. L.,
96, 109
Novikoff,
A.
B.,
291,
328
Nowell,
P.
C., 179, 198, 212, 220, 247,
Noyes, W. D., 256, 266
Nunes, Petisca,
J. L.,
317,
322
Nussenzweig, V., 4, 55, 97, 112, 278, 280,
Nyhorg, W. L., 136, 195
255,
265,266,269,
316,
328
286,
289,
322, 324,
372,
379
0
Oberdorfer, R., 343,
378
O’Connor, J. M., 60, 96, 113
Odartchenko, N., 208,
263
Odeblad,
A.
M., 288, 328
Odehlad,
E.,
288,
328
Odell, T. T., 255, 263
O’Donnell,
I.
J., 80,
114
Oghurn,
C.
A.,
124, 167,
195
Ogden,
D. A, , 258, 261
O’Gorman,
P.,
183, 194
Ohm, J., 28,
54
Ojeda,
A.,
175, 196
Oknzaki, W.,
117,
193
O’Konski, C.
T.,
345,
349,
376, 378
Old, L. J.,
128, 150, 156, 186,
192, 193,
Olins,
D. E.,
371, 372,
377, 379
Ollier, M.
P.,
97,
11
1
Oncley, J. L., 342, 345, 379
O’Neill,
J.
O., 120, 192
Onoue, K., 59, 64, 74, 84, 86, 87, 89, 91,
92, 112, 114, 355, 371,
372,379,
380
Oort, J. O., 250, 269, 312, 323
301, 330
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
http://slidepdf.com/reader/full/advances-in-immunology-vol-12-f-dixon-h-kunkel-ap-1970-ww 419/430
400 AUTHOR INDEX
Oppenheim, J.
J.,
179,
198,
215,
266,
286,
Orange,
R.
P., 308, 309,
326, 328
Orbegoso,
C .
M . ,
317,
323
Orfei,
E.,
317,
327
Orlans,
E.,
103,
114
Orloff,
M. J. ,
172, 173, 195, 199
Orme-Roselli,
L.,
317,
329
Osboa,
D.,
173,
197
Osmond,
D. G.,
208, 209, 212,
266, 267,
270
Osoba,
D.,
202, 226, 232, 248,
265, 266,
271, 273,
281
Osserman,
E.
F.,
46,
55
Osterland,
C. K.,
12, 17, 18, 19, 44, 48,
53, 54, 55,
71, 72, 98,
109, 377
Oth, A,, 145,
193
Ottinger,
B.,
287,
324
Oudin,
J.,
6, 7, 11, 25, 52,
55, 56,
150,
Ovary, Z., 276,
279
Owen,
E.
R., 166, 172,
198
Owen,
J.
J.
T.,
212,
265
Owen,
R.
D.,
170,
198,
235, 255,
266
Ozer, J. H. , 156,
198
328
198
P
Padawer, J., 307, 328
Page,
L. B.,
40,
54
Pain, R. H., 335, 336, 380
Paletta,
B., 343, 378
Palm, J., 117, 118, 123, 124, 146, 163,
Palmer, J.
L.,
370,
380
Panayi,
G . S.,
207,
261
Papermaster, A. M . , 136, 198
Papermaster,
B. W.,
103, 104, 106,
111,
115, 119, 136, 142, 145,
183,
188,
198, 199,
202, 205, 206, 213, 229,
238, 262, 266, 380
Pappenheimer, A.
M.,
Jr., 29, 30,
55,
56,
85, 114, 136, 168, 198, 200
Pardee,
A.
B., 118,
198
Parish,
C.
R., 287, 328
Parish,
W.
E., 313,
329
Park, B., 313, 326
Parker,
D. C.,
33, 50, 55,
56
Parker,
J.
W., 313,
329
Parker, S. J., 199
166, 182,
194, 197, 198
Parkes, A. S., 168, 198
Parkhouse,
R. M . E.,
67, 68, 69, 70, 75,
Parks,
E.,
291,
323
Parks, J.
J.,
271,
279
Parrott,
D. M . V.,
225, 234,
262, 266
Patrick,
R.,
286, 289, 322
Patt,
H. M . ,
208,
264
Patterson,
R.,
307, 313, 329
Paul, W.
E.,
96,
114,
217,
266,
276,
280
Payne,
L.
N. , 117,
195
Payne,
R.,
183, 186,
192, 198
Peacoke,
N.,
182,
191
Pearmain,
G.
E.,
179,
197,
316,
329
Pearsall,
N .
N. , 221, 266
Pearse,
A. G. E.,
301,
325
Pease, G. L., 256,
265
Pedersen,
K.
O.,
58,
111, 378, 380
Pegrum,
G. D.,
209,
266
Pellegrino,
M.,
119, 136, 142, 145, 146,
Pepe,
F. A, ,
290, 293, 295,
323
Pepper,
D. J. ,
147, 198
Perchalski, J.
E.,
88,
114
Perey, D. Y . , 272, 280
Perkins,
E. H.,
231, 244,
266,
291,
329
Perlman, R.
L.,
66,
113, 114,
351,
379
Perlmann,
H.,
189,
198
Perlmann, P., 185, 189,
195, 196, 198
Pernis,
B., 7, 55,
99, 101,
114,
290,
329
Perraniant,
F.,
173,
197
Perry,
V.
P., 235,
264
Persijn, J. P., 292, 294,
323
Petermann,
M. L.,
85,
114
Peters,
R.
S . ,
165,
195
Peterson,
J.
K. ,
183,
194
Peterson, P. A., 349, 378
Peterson,
R. D . A.,
226,
261,
272,
279,
280
Pethica,
B . A.,
93,
108
Pflumm,
M. N. , 81,109
Phelps,
H. J.,
183,
198
Phillips, J. H., 256, 268
Phillips,
M . T.,
307,
327
Phillips, R .
A.,
204, 208, 231, 268
Phillips,
R. J . S.,
255,
259
Pierce, C . W. , 218, 229, 237, 239, 266,
Pierce, J. A., 276, 279
Pike, R.
M . , 60,
4,
114
100,
102,
114, 259,
355,
380
177, 184, 188, 196, 198, 200
286,
329
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
http://slidepdf.com/reader/full/advances-in-immunology-vol-12-f-dixon-h-kunkel-ap-1970-ww 420/430
AUTHOR INDEX 401
Pilz,
I.,
343, 349, 351,
378, 380
Pinchuck, P., 218, 266, 277, 281, 304, 329
Pincns,
J .
H. ,
12, 29, 30,
56
Pink,
J.
R. L., 79, 80, 82, 87, 110, 114
Pinkett,
M. O.,
212, 220,
266
Pinnas, J. L., 273, 281
Pinnell, S. R., 349,
381
Pinniger, J. L., 288, 329
Pisciotta, A .
V.,
204,
266
Plaut, A .
G . ,
84, 87, 114
Playfair,
J. H. L.,
205, 206, 229, 238, 252,
266, 278, 281
Plescia, 0 .
J.,
277, 281
Plotz,
P.
H.,
95, 98,
114,
216,
266
Plymin,
G.,
289,
322
Pogo, B. G. T., 317, 329
Pokovna, Z., 168, 177,
193
Polani, P. E., 256, 267
Poljak, R.
J.,
356, 357,
377, 380
Pollard, B., 171, 195
Polley,
M . J.,
220,
262
Polyanovsky, 0. L., 346, 380
Popp,
D. M.,
156,198
Popp, R.
A.,
156,
198,
235, 255,
266
Poratli, J., 33,
53,
55,
56
Porod, G . , 343, 378
Porter, D. D., 38,
56, 266
Porter, K.
A.,
210, 262
Portei, K. R., 314,
329
Porter,
R.
R., 1, 2, 53, 54,
56,
80, 114,
334, 363,
377, 380
Potter, M., 41, 43, 44, 46, 47, 54, 56, 377,
379
Poulik, M. D., 1, 53
Prahl,
J .
W.,
6, 26,
56
Prendergast, R. A , , 251, 266, 339, 378,
Prescott, B., 287, 324
Press,
E. M.,
1,
54,
59, 81, 82,
109, 114,
363, 376, 377
Pressman, B. C. , 283, 305,
324
Pressman, D., 29, 41,
55,
56, 64, 9, 91,
92, 100,
114, 115,
355, 371, 372,
379, 380
381
Preston,
F.
W., 183,
199
Pribnow, J. F., 244, 266, 274, 282, 286,
303,
329
Price, G . , 271, 281
Priore, R. L., 205, 206, 224, 230, 231,
260, 267
Prose, P., 311, 31.3,
325
Pruzanski,
J.
J., 307, 313, 329
Piichwein,
C . ,
343, 349, 351,
380
l'iilznik, U.
H.,
205, 266
Putnam,
F. W.,
43,
56,
GO 64, 76, 77,
78, 79, 80, 81, 83, 112, 114, 116
Pye,
J.,
254,
265,
288, 289,
322, 328
Q
Qiiagliata, F., 321, 330
Quaglino,
D.,
207,
260
Qunstel, J.
H. ,
183, 192
Qtiastt. , M . R., 321,
329
Quie,
P.
G.,
310,
329
Quinn,
I,. Y.,
301,
328
R
Rabinovitch, M., 97, 114 , 291, 329
Rabson, A.
S.,
207,
261
Hadenin, H.,
88,
112
Rndl,
J.,
256,
260
Radovich,
J.,
224, 237, 239, 241, 242,
243, 246,
266, 268,
271, 272, 273,
276, 278, 282
Radzimski, G . , 372,
380
Raidt,
D.
J., 214, 224, 229, 234, 266, 271,
Majewski, K., 277, 282
Ramseier, H., 178, 189,
198,
285, 301,
Hanadive,
N.,
313,
329
Rao, C. V.
H. ,
277, 281
Rapnport,
F. T.,
119, 135, 150, 152, 160,
Rapp,
H .
J.,
64,
95, 96,
108,
109,
111
Ratclifl', P., 61, 114
Ravn,
H.,
220,
266,
292, 293, 295,
329
Ray,
H.
E. M., 272, 278, 279
Raymond,
J.,
137,
198
Raymond,
M.,
363,
379
Hazafitnahaleo, E., 73,
108
Reade,
P. C. ,
301,
327, 329
Ready, D., 209, 266
Reed, W. D., 29, 55
Rees,
R.
J.
W., 301,
322
Reichlin, M., 63, 84, 86, 109, 353, 376
Reinert-Wenk, U., 146, 147, 169, 196
Reisfeld,
R. A., 4,
0,
56,
119, 136, 137,
138, 140, 141, 142, 144, 145, 146,
282
330
193, 198
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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402
AUTHOR
INDEX
149, 154, 155, 162, 166, 174, 177,
178, 179, 180, 188, 196, 198
Reisler, E.,
380
Reisner, C . A., 74, 75, 114
Reiter, I. H., 210, 266
Rekers,
P. E.,
255, 266
Remold, H.
G . ,
316, 330
Resnitzky,
P.,
226, 268
Rhodes,
J.
M. , 218, 220, 258, 266, 287,
Rice,
S . A.,
46, 53
Rich,
N.
R., 185, 200
Richards, F. F., 40, 54, 363, 372, 378
Richter,
M.,
207, 209, 210, 211, 214, 215,
216, 217, 220, 221, 223, 224, 225,
226, 227, 228, 233, 235, 236, 237,
239,
240,
243, 244, 245, 246, 247,
252, 254, 255,
258, 260, 263,
265,
266, 267
292, 293, 295, 304, 322, 329
Riddle, J. M., 305, 329
Rieder, R. F., 11, 56
Rigler, R., 319, 320, 327, 329
Riha,
I , ,
205, 268, 290, 326
Rimon,
A,,
88,
114
Ringertz, N. R., 319, 324
Rippin,
A,,
170, 200
Rittenberg, M. B., 277, 282
Roan,
P.
L., 255, 265
Roane,
P.
R., 183, 199
Robbins,
E.,
317, 329
Robbins, J. B., 32,
56,
60, 94, 114, 376
Robbins, J.
H.,
286, 327
Robert, B., 88, 114
Robert,
W.,
273, 278, 282
Roberts,
A.
N.,
290,
329
Robineaux, R., 317,
325, 329
Robins, M . M., 256, 266
Rockey, J.
H.,
59, 93, 112, 336, 338, 339,
340, 358, 359, 360, 371, 372, 377,
379, 380
Rodey, G.,
313,
326
Rodkey, L. S., 26, 56
Roelants,
C. E.,
219, 267
Rogentine, G.
N. ,
119, 131 , 132, 133, 135,
147, 148, 150, 151, 152, 153, 155,
197, 215, 266
Roholt,
0. A.,
41,
56,
371, 372, 380
Roitt, I. M., 105, 111, 215, 246, 254, 262,
Roizman, B., 183, 199
264
Rose,
B.,
210, 211, 223, 233, 239, 245,
Rose,
M.
E.,
103,
114
Roseman,
J.
H., 267, 271, 273, 282, 286,
Roseman, W., 293, 296, 300, 325
Rosen, F.
S.,
272, 278, 279, 280, 282
Rosenau, W., 208, 267
Rosenberg,
I.
T., 161, 197
Rosenfield, H.
A.,
173, 199
Rosenheck, K., 362, 380
Ross, D. L., 358, 359, 362, 380
Ross, M.
H.,
311, 313, 325
Rosse,
C.,
208, 212,
265
Rossi,
C.,
356, 380
Rossi, T., 101,
115
Rostenberg, I., 99, 115
Rothberg, R., 273, 278, 282
Rothfield, N.
F.,
88, 114
Rotman, B., 183, 184, 193, 196, 199
Rouillard, L.
M.,
206, 260
Rowe, A. J., 347, 377
Rowe, D.
S.,
60, 114, 340, 380
Rowley,
D.
A,,
202, 221,
264, 267,
273,
Rowsell,
H.
C., 312, 328
Ruddle,
N.
H., 178, 199
Ruffilli,
A,,
6, 53, 373, 380
Ruoslahti, E., 89, 105, 112, 202, 264
Ruskiewicz, M., 123, 159, 162, 178, 192,
Russell,
P.
S.,
159, 161, 162, 163, 165,
Russell, W.
J.,
103, 104, 116
Ruth, R.
F., 267
Rutishauser, U., 79, 80, 81, 109, 110,
Rychlikova, M., 177, 193
Rymo, L., 376
258, 266
329
281, 282, 285, 301, 329
196
172, 198
337, 338, 343, 377
s
Sabet, T., 219, 267
Sabin, F. R., 288,
329
Sachs, L., 205, 266
Sado, T., 271,
281
Sage, H. J., 277, 280
Sahler, 0. D., 256, 268
St. Rose, J.
E.
M., 277, 279, 282
Sainte-Marie, G., 203, 235, 267
Saito,
K.,
301, 329
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
http://slidepdf.com/reader/full/advances-in-immunology-vol-12-f-dixon-h-kunkel-ap-1970-ww 422/430
AUTHOR INDEX
403
Salmon,
S.,
94, 95,
111
Salsbury,
A .
J.,
93,
114
Salvin,
S. B. ,
168, 199, 200, 276, 282
Samaille, J., 59, 108
Sampson, D., 306,
329
Saniuelson, I. K., 251, 263
Sandberg, A.
L.,
95, 96,
115
Sanders, B. G., 6, 50, 54
Sanders,
C. L.,
292, 294,
329
Sanderson,
A.
R., 118, 119, 129, 130, 132,
134, 135, 143, 148, 150, 151, 152,
153, 156, 185, 187, I 9 9
Sandor, G.,
88, 114
Santamaria,
A.,
311, 325
Santos-Buch, C .
A.,
313,
329
Sarkar,
P.,
380
Sater,
J.,
313,
326
Saunders, G. C., 207,267
Sawant, P.
L.,
305,
330
Scatcbard,
G.,
342, 379
Schachnian,
H. K.,
337,
380
Schaeffer,
S.,
306, 327
Schapiro,
H. C.,
60,
109
Schechter,
B. ,
275, 282
Schechter,
I. ,
89,
111 ,
275,
282,
288, 289,
290, 326, 327
Scheer, S . C., 235,
259
Scheinberg, I. H., 73, 113
Schellekens, P. T . A,, 179,
194
Scheraga, H. A., 335, 380
Scherer,
J.,
305, 306, 311,
326, 327
Schilf, F., 210, 267
Schlossman,
S.
F., 5,
56
Schmale, J.,
87,
101, 114, 116
Schmitz,
P.
J.,
343, 378
Schneider, W . C., 136, 195
Schoenberg,
M . D.,
244,
267
Scholtan, W., 62, 112, 351, 378
Schooley,
J.
C. , 208, 267
Schreibman,
R.
R., 179, 195
Schrohenloher,
R . E. ,
78,
87,
90, 91,
108,
Schubert, D., 44, 56
Schuit, H . R. E., 101, 116, 210, 269
Schulenburg,
E. P.,
44, 45, 54
Schulman, S., 89, 111
Schultze, H. E., 59, 63, 64, 1 1 1 , 114
Schwartz,
H. J.,
292, 295, 302,
323
Schwartz,
I. R.,
256,
258
1 1 1 ,
114
Schwartz,
R. S., 163,
173,
191, 199,
252,
Schwarzenberg,
L.,
256, 264
Schwartzman, J. S., 229, 267
Schwartzman, R.
M., 380
Scothorne,
R.
J., 199
Scudeller,
G.,
117, 150, 152, 184,
193,
Seegers,
W.,
309,
329
Segal, S. , 216, 267
Segre, D., 99,
115
Segre, M., 99, 1 1 5
Sehon, A .
H.,
40,
54
Seifert,
L.
N.,
173, 199
Sekora, H., 343,
378
Sela, M., 4, 32,
56,
60, 9, 94, 111, 112,
114,
171, 189,
195, 197,
275,
282,
287, 288, 289, 290, 326, 327, 367,
368,
369,
377, 378, 379
Seligmann, M.,
68,
84, 86, 87, 113, 115,
Sell, K. W., 206, 263
Sell,
S.,
99, 115, 202, 207, 215, 228, 231,
267, 316, 329
Seller,
M . J . .
256,
267
Selner, J. C. , 236, 238, 259, 260, 271, 279
Selye,
H.,
307,
329
Seon, B-K., 41, 56
Sercarz, E . E., 185,
199,
213, 214, 231,
Serra,
A.,
117, 150, 152,
193
Sessa, G., 315, 329
Shaher,
A.,
293, 296,
322
Shand,
W. S.,
93,
114
Shapiro,
F.
172,
199
Shapiro,
J.,
58,
115
Shapiro,
M .
B., 71, 78,
113
Sharp,
J. A.,
244,
267
Shearer,
G .
M., 205, 206, 208, 209, 214,
Shekarchi, I. C., 210,
261
Shellam,
G. R.,
225, 228, 252,
258,
273,
Shelton,
E.,
68,
70,
102,
115
Sheridan, S., 267
Shibko,
S. ,
305,
330
Shimada, A., 119, 130, 131, 132, 133,
134, 135, 146, 147, 148,
151,
152,
153, 154, 155, 199
Shimizu,
A.,
80,
81, 112
264
200
353,
379
251, 253, 259, 260, 267
224, 230, 231,
260, 267
279
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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404 AUTHOR INDEX
Shin, H. S., 96,
115
Shinoda, T., 64, 78, 79, 80, 81, 83, 116
Shortman,
K.,
234, 247,
262, 267
Shortnian, K. D., 228,
265
Shreffler, D. C., 117, 118, 194, 199
Shrek, R., 183,
199
Shulrnan,
S.,
63, 115
Sieniienowicz,
R.,
204,
259
Sigel, M. M., 103, 104, 109, 116
Silverman,
L.,
156,
194
Silverman, M . S . , 168, 192, 244, 266, 274,
282,
286, 303,
329
Silvers,
W.
K., 117, 195, 248, 249, 259
Silverstein,
A. M.,
106,
115,
179,
199,
202, 268
Siminovitcfi, L., 204, 205, 206, 208, 210,
214, 228, 231, 263, 267, 268, 269,
270
Simmons, E. L., 255, 263
Simmons,
R.
L., 248,
267
Simmons, T., 180, 197
Simnis, E.
S.,
44, 45, 54,
377
Simons, M. J., 219, 267, 279, 282
Simonsen,
M.,
167,
199,
247, 256,
265,
267, 279, 282
Sinclair, N.
R.,
225,
267
Singer, S . J., 42, 50, 53, 56, 83, 110, 363,
379, 380
Singhal, S . K., 209, 210, 215, 216, 217,
223, 227, 228, 233, 236, 239,
266,
267
Siskind, G.
W.,
5,
53,
105, 115, 168,
199,
202, 213, 214, 217, 266, 267, 287,
326
Sjodin,
K.,
248,
260
Skaniene, E., 215,
267
Slater, F. D., 226, 262
Slobin, L. I., 380
S h e , D., 166, 172, 198
Small,
h4.,
226, 243, 268
Small,
P. A.,
Jr., 4, 20,
56,
63, 75, 76, 77,
78, 88, 102, 103, 104,
109, 112,
114,
335, 336, 337, 340, 356, 376, 370,
380
Smith, F.
W.,
136,
199
Smith, J. M., 171, 172, 196, 197, 199
Smith,
L.
G., 248, 269
Smith, R. F., 168, 199, 276, 282
Smith,
R. T.,
171,
199,
202, 254,
268
Smith, S. B., 252, 263
Smith, W.
W.,
205,261
Smithies,
O.,
205,
259
Smyth, D. S., 344,
380
Snell, C.
P.,
196
Snell, G. D., 117, 121, 162, 168, 189,
194, 195, 199
Snigurowicz, J., 77, 116
Snyderman, R., 96, 109, 115
Solheim, B. G., 93, 99, 111
Solomon, A., 77, 88, 89, 115, 349, 380,
Solomon, J. M., 287, 304, 325
Soothill,
J.
F.,
61,
114
Sorkin, E., 305, 315, 322
Sosin,
H.,
249,
268
South, M.
A.,
272, 280, 380
Southworth,
J.
G., 136, 137, 178, 179,
Sparrow,
E.
M., 160, 163, 165,
192, 199
Spector,
W.
G., 211, 250, 268
Spencer,
R.
A., 118, 183,
191, 199
Spilberg,
I.,
305, 306, 311, 330
Spiro,
D.,
292, 294,
331
Spitznagel, J. K., 306, 313, 325, 326, 331
Spooner,
R.
L., 206,
263
Spragg,
B.
P., 72, 73,
115
Stanworth, D. R., 59, 61,
114,
115,
379
Staples, P.
J.,
252, 268
Stark, J . M., 289, 330
Stark, O., 117, 199
Starshinova,
V.,
94, 109
Stastny,
P.,
207, 269
Stavitsky,
A .
B.,
202,
268,
287,
304,
325
Stechschnlte,
D. J.,
308, 309, 328
Steinberg,
A.
G., 99, 115
Steinberg, I. Z., 369,
379
Steiner, L. A., 65,
115,
358,
359, 380
Steiner,
R.
F., 66,
115,
344,
381
Steinmuller, D., 165,
199
Sternlieh, I., 73, 113
Sterzl, J., 106, 115, 202, 205, 268, 271,
Stetson,
C.
A.,
164, 168, 186,
195, 199
Stevenson, C .
T.,
363, 364, 365, 371, 374,
Stielini,
E. R.,
85, 101, 110, 115
Stiffel,
C.,
206,
217, 259
381
196
282
381
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR
INDEX
405
Stimpfling,
J. H.,
117, 121, 146, 148, 168,
Stoho,
J.
P.,
88,
115
Stockert, E., 128, 150, 156, 186,
192, 193
Stollar,
B. D.,
95, 96,
109, 115
Stoloff, I. L., 210,
268
Stone, M. J., GO,
66, 79, 80, 83, 86, 90,
94,
113, 115,
355,
381
Stone,
S . H.,
175,
199,
202,
268
Stoner, R. D., 210,
268
Storb,
U.,
206,
268
Straw, D. L., 287, 304,
324
Straw,
W.,
291, 292, 295, 299,
329
Streihlein,
J .
W.,
178,
198
Strober,
S .
203, 223, 229,
268,
286,
329
Strober,
W.,
101,
116
Stryer, L., 66,
116,
346, 356.
381
Stuart, F . P., 170,
200
Shimpf,
P. R.,
136,
199
Stiltman, O., 229, 247, 248, 249, 257,
Sulitzeami,
B.
D., 38,
54,
106,
113, 114,
Suinmerell,
J .
M . ,
119, 120,
192, 199
Sundberg, L., 33, -55
Suran, A.
A., 103, 104,
1 1 5
Sussdorf,
D.
H., 229,
268
Suter, E., 94, 114, 385, 301,
329, 330
Snznki, T., 61, 62, 63, 74, 75, 76, 77, 78,
Svehag, S. E., 59, 68, 70,
109,
115, 352,
Svejgaard, A , ,
150,
152,
196
Swedlund,
H. A.,
75, 88,
109, 110, 1 1 5
Sword,
C.
P.,
293, 298,
322
Syeklocha,
D.,
206, 228,
268
Szakal,
A.
K., 289,
326
Szenllerg, A., 100,
114,
205, 247,
267,
268,
272, 279,
282
Szonyi,
L.,
210,
263
169,
196,
199
264, 268
216, 246,
265, 268
82, 84, 86,
115,
351,
381
376, 381
T
Tada, T., 95, 111
Taichman,
N . S.,
312,
328, 330
Tailor,
E.,
162,
199
Takahashi,
M.,
100,
1 1 5
Takaniizawa,
A, , 96,
11
1
Takatsuki, K., 46,
55
Takeuchi,
Y.,
309,
327
Tala],
N.,
95, 98,
114
Taliaferro,
L. G.,
229, 244,
268
Taliaferro, W.
H.,
101,
115,
173, 199,
229, 244,
268
Talmage, D. W.,
6, 54,
77, 99, 101,
111,
115,
224, 237, 239, 241, 242, 243,
246, 266, 268,
271, 272, 273, 274,
276, 278,
280, 282
Tan, E. M., 349,
381
Tanaka, J., 362,
378
Tanford,
C.,
65, 110, 335, 337, 341, 342,
343, 350, 358, 359, 360, 363, 364,
365,
366, 367, 370, 373, 375,
376,
377, 378, 379, 381
Tannigaki,
N.,
100,
115
Tappel,
A. L., 305,324, 330
Tarail,
M. H.,
103, 104,
115
Taranta,
A.,
321,
330
Taubinan,
S .
B., 305, 315,
330
Tausche, F.
G.,
255,
263
Taylor,
A .
C.,
164,
200
Taylor, P., 183,
200
Taylor,
R. B.,
203, 232, 223, 225, 227,
228, 229, 233, 237, 238, 239, 252,
268,
278,
282,
285,
330
Teagrie, P.
O.,
229, 248,
268
Tenipete-Caillourclet,
M.,
78, 110,
377
Temple,
N.,
220,
263
Terasaki, P.
I.,
176, 183, 185, 186,
192,
Terrinha,
A.
M., 317,
322
Terry,
E. W .,
59,
110
Terry,
R.
W., 306, 307,
330
Terry,
W. D.,
80, 81, 83,
112, 113,
215,
Thaxter,
T.
H.,
165,
195
Thoenes,
G .
H.,
115
Thomas, E. D., 235, 256,
2.59, 268
Thomas, L.,
160,
163, 168,
191, 198, 199,
Thompson, E., 209,
266
Thorbecke, G.
J., 210,
240, 248, 254,
260,
Thorp,
N. O., 380
Till,
J.
E., 204, 205, 206, 208, 210, 214,
228,
231, 259, 263, 267, 268, 269,
270
200,
226, 256, 257,
264
266,
356,
381
313,
330
261,
285, 286, 301,
325, 330
Tiselius,
A , , 378
Tjio, J. H , 286,
327
Todd,
C.
W., 6, 24, 25, 26, 36,
53, 54,
56, 99,
115
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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406 AUTHOR
INDEX
Toepfer, J.
R.,
208,
265
Toivanen, P., 101,
115
Tolone,
G.,
97,
109
Tomasi,
T. B.,
Jr., 59, 63, 84, 86, 87, 88,
90, 93,
109, 112, 114,
115, 353,
376, 381
Tooney, N. M.,
376
Torii, M., 48,
56
Torrigiani,
G.,
99, 101, 105, 111, 114,
Tosi, R. M., 184, 193,
200
Trainin, N., 226, 243,
268
Treadwell, P. E., 313,
329, 330
Trentin,
J.
J., 209, 213, 225, 230, 231,
Tridente, G., 247, 248,
260
Triplett, R.
F.,
214, 229, 237,
238, 259,
Tripp, M., 184,
192
Troll, W.,
319,
320, 321,
326
Troup, G. M., 183,
198, 200
Trump,
C.
N., 42, 56
Tumerman,
L.
A., 345, 346,
380, 381
Turk, J.
L.,
2-50,
269
Turner,
M.
W ., 349,
381
Turner,
R.
W. A., 204,269
Tyan, M.
L.,
208, 209, 249,
269
215, 246,
262
260,
269
271, 273,
279
U
Udin, B., 43,
56
Uhr, J.
W.,
88, 97, 105, 106, 110, 115,
116, 168,
200, 202, 269,
292, 293,
294, 295, 296, 297, 298, 300, 303,
330
Umiel, T., 243,
269
Unanue, E. R., 218, 220, 244,
269,
287,
293, 295, 296, 300, 302, 303,
322,
330
Underdown,
B. J.,
44, 45,
54
Ungar-Waron,
H.,
89, 112
Uphoff, D. E., 253, 255, 256, 263, 269
Upton, A. C., 208, 209,
260
Uridiara , T., 306, 312,
323, 328
Urnes, P., 357,
381
Urrmti, J., 101, 110
Urso,
I .
S. ,
226, 261
Utsunii, S., 344, 363,
380, 381
Uycki , E.
hl . , 321,
330
V
Vaerman, C.,
381
Vaerman, J.-P,, 339,
381
Vaes, G.,
330
Valentine,
F.
T., 207,
269,
316,
330
Valentine, R. C., 347, 348,
381
Vallotton, M., 40, 54
Van Alten,
P.
J., 272,
279, 281
van Bekkum, D. W., 247, 248, 256,
260
Van Der Muel, V. A., 206,
270
Van der Schaaf, D.
C.,
59, 113
Van de Water, L., 286,
327
Van Eyk,
H . G.,
349,
381
van Furth,
R.,
101,
116,
210, 215, 220,
Van Holde,
K. E.,
370,
380
van Hooft, J. I. M., 247, 256,
260
Van Leeuwen, A., 117, 123, 148, 186,
Van Leeuwen,
G.,
75,
112
van Loghem, J. J.,
194
Van Meter, R., 272,
282
Vann, D. C., 245,
269
Vannier,
W. E.,
64, 95,
I l l
Vannotti,
A , ,
254,
261
Van Orden, H. O., 8,
56
van Rood, J. J., 117, 118, 123, 148, 186,
Van Twisk, M. J., 206,
270
Van Zwet, T.
L.,
88,
112
Vas, M. R., 179,
192,
207,
258
Vas,
S.
I., 183,
192
Vassalli, P., 278,
280,
289,
323
Vaughan,
J.
H., 8 8 , 1 1 0
Vaughn,
R. B.,
291,
330
Vazques, J. J., 277,
280
Verbo, S., 179,
195
Vernon-Roberts,
B.,
212, 221, 269
Vice, J. L., 6,
56
VigCio, J.
D.,
317,
322
Vinton, J. E., 71, 78,
113
Virolainen, M., 211, 212, 220, 266, 269
Vischer, T. L., 207, 269
Vim,
D.
C., 128, 180,
193, 200
Voisin,
G.
A., 168, 169,
200
Volini,
F. I.,
317,
327
Volkman, A., 211, 212, 220, 250,
269
Voss, E. W.,
Jr.,
89, 92, 103, 104, 110,
221,
26*9
193, 200
193,
200,
256,
260,
296,
325
116
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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AUTHOR INDEX
407
Vredevoe, D., 183,
192
Vurek, G. G., 183, 194
W
Wahl, P., 345, 346,
381
Waite, J. B., 177, 197
Waksnian,
B. H.,
178,
199,
207, 249, 250,
Walburg, H. E., Jr., 289, 291,
326, 329
Waldenstriim,
J.,
381
Waldmann,
T. A.,
101,
108, 116
WaIford,
R.
L., 150, 152, 169, 176, 183,
193, 195, 198,20 0
Walker,
R.
I.,
306,
326
Wallace, J.
H.,
183,
195
Wallach, D.
F.
H., 155, 200
Wallis, V., 204, 214, 222,
228,
233, 237,
238, 251, 252, 260, 262, 271, 273,
280
251, 252, 259, 263, 268, 272, 280
Walters, N. I., 211, 268
Ward, P. A., 96, 116, 305, 315, 316, 326,
Warner,
N.
L., 95, 98, 106,
108, 112,
Warren, S. L., 255, 266
Wasaka, M., 316, 329
Wasson, D.,
170,
200
Waterfield, M. D., 29, 56
Waterson, A. P., 70, 108
Waterstone, D. J., 166, 172, 198
Wattiaux, R., 283, 284, 292, 294, 305,
Waxdal, M. J., 79,
80,
81, 110, 337, 338,
Weber, G., 66,
112, 116,
345, 346,
381
Weigle, W. O., 251,
269,
276,
281, 282,
Weiler, E., 285, 330
Weiler, 1. J., 285, 330
Weiner, L. G., 165, 199
Weintranb, M., 137, 198
Weisberger,
A.
S .
244, 267
Weiser, R.
S .
206, 221,
262, 266, 268,
Weiss,
D.,
209, 225, 230, 231,
268
Weiss, L., 303, 317, 324, 330
Weiss, N . S. 271,
281
Weissman, I. L., 235, 237, 269
Weissmann,
G.,
284, 290, 292, 293, 294,
295, 296, 297, 298, 300, 303, 305,
330
248,
269,
272, 279,
282
324, 330
343,
377
285, 305, 306, 324, 330
269
306,
307,
308,
310, 311,
313, 314,
315, 316, 317, 318, 319, 320, 321,
323, 324, 326, 327, 329, 330
Wellensiek, H. J., 290,
330
Welscher, H. D.,
340, 380
Weltman, J. K., 345, 346, 381
Weston, P. D., 310,
330
Wetter, O., 65, 116
Wetzel, B.,
164, 194
Weyzen, W. W. H., 219, 263
Wheeler,
H.
B., 172,
198
Whitby, J. L., 210, 262
White,
A.,
226,
262, 263
White,
J.,
307, 313,
323
White, R. G., 173,
200,
202, 235, 250,
265, 289, 325, 330
Whitney,
P.
L., 366, 367, 370,
376, 381
Whyatt, J. P., 212, 259
Wiadrowski, M. N., 235,
264
Wbo, M., 292, 294, 330
Wide, L.,
378
Wiedermann,
G.,
316, 330
Wiener, E., 291, 294, 301,
323
Wiener, J., 292, 294,
331
Wigzell,
H.,
185, 186,
200,
206, 214, 215,
216, 217, 220, 230, 231, 246, 259,
267, 269,
275,
281, 282
Wikler, M., 64,
7 8 ,
79, 80, 81, 83, 116
Wilkinson,
J.
M., 6,
56,
99,
116
Wilkinson,
P.
C., 173, 200, 289, 325
Willard,
H.
G., 248,
269
Williams,
G.
M., 289, 290, 292, 293, 295,
298, 300,
322, 328, 331
Williams, R.
C.,
Jr., 7, 10, 55, 56, 75,
100,
116,
349,
381
Williamson,
A.,
5 , 53
Willonghhy,
D.
A., 211,
268
Wilson, A. B., 215, 260
Wilson, D. B., 202, 247,
269,
286,
331
Wilson,
P.
E., 159, 197
Wilson, R. E., 136,
170, 193, 200
Windelstein, M., 242, 260
Winfield, J. S. 293, 296, 300,
325
Winkler, M. H., 344, 381
Winn,
H.
J.,
199
Wintiohe, M. M., 208, 269
Wisse, E. , 290, 294,
324
Wissler, R. W., 271, 282, 288, 289, 326
Witebsky, E., 182,
191
Wochner,
R.
D., 101, 108
7/24/2019 Advances in Immunology [Vol 12] - F. Dixon, H. Kunkel (AP, 1970) WW
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408
AUTHOR INDEX
Woessner, J.
F., 306,
331
Wofsy, L., 50,
56
Wolf,
N.
S.,
209, 213, 225, 230, 231,
260, 269
Wolf,
S .
M., 248, 267
WoIfe, H. R., 272,
279
Wollheim, F. A,, 75, 77, 116, 380
Wolstencroft, R. A., 179,
198,
207,
261,
Wood, M. L., 159, 161, 162, 163, 165,
Woodruff, J., 235, 269
Woodruff, M. F .
A.,
173, 200
Woods, P.
A.,
235,
265
Woods, R., 77, 116
Wortis, H.
H.,
205, 225, 228, 239, 260,
Wright, S., 175, 200
Wu, A. M. , 204, 208, 210, 231, 270
316, 327
172, 198
268
Y
Yagi, Y., 64, 89, 91, 92, 100, 114, 115,
Yakulis, V., 87, 92, 101, 109, 114, 116
Yamamura,
Y.,
59, 74, 84, 86, 87, 112,
Yang, J. T., 65, 113, 357, 362, 379, 381
Yguerabide, J., 66, 116, 346, 381
Yoffey, J. M., 208, 209, 212, 213, 262,
355, 379
114
265, 270
Yonemasu, K., 96, 111
Yoo,
T.
J., 363,
381
Yoshimura,
M.,
277,
279, 282
Young,
D .
A,, 314,
327
Yount, W . J., 5, 22, 53,
56,
339, 378
Yphantis,
D.
A.,
335,
337, 381
Yunis, E. J., 229, 248, 268, 270
Z
Zaalberg, 0.B., 206, 270
Zagyansky, Y.
A,,
345, 346,
380, 381
Zajtchuk, R., 136, 200
Zak, S .
J.,
277,
281
Zaleski, M., 247, 265
Zappacosta, S., 41,
S5,
356, 372,
379, 381
Zarlengo, 358, 364, 373, 377
Zeligs,
J. D.,
305,
326
Zetterberg, A,, 320, 331
Zeya, H. I., 306,326,331
Ziff, M., 305, 324
Zigelbaum, S., 381
Zimmerman, C . E., 170, 200
Zinder,
N.
D.,
371, 372,
377
Zinneman, H. H., 73, 111
Zmijewski, C., 83,
200
Zullo, D.
M . ,
26,
56
Zumpft,
M., 191
Zweifach,
B.
W., 309, 311, 313,
324, 327
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A
Alloantigenic determinants, see also
Transplantation antigens
chemical nature, 145-150
physical nature, 150-157
Amino acid(s ) , sequence of immuno-
Angiotensin, antibodies to, 40-41
Antibodies, see also Globulins, Immuno-
globulins, 8-10
globulins, Macroglobulins
angiotensin, 4 0 4 1
bacterial carbohydrates, isolation from
antisera, 30-37
combining sites, heterogeneity, 5-6
cytotoxic, transplantation antigens and,
human, restricted heterogeneity of,
molecular uniformity, discussion, 48-
myeloma proteins, 43-47
myoglobin, 4 0 4 1
paraproteins, 4 3 4 7
passively administered, antibody syn-
thesis and, 277
pneumococcal capsular polysaccha-
rides, 29-30
properties, limited heterogeneity and,
restricted heterogeneity
183-189
10-12
53
3-10
experimental generation of, 12-43
factors influencing occurrence, 37-
40
streptococcal carbohydrates, 12-28
synthesis
adherent cells and, 273-275
antigenic competition and, 276
macroglobulins and, 106
multiple antigenic determinants and,
passively administered antibody and,
276-277
277
synthetic antigens, 41-43
Antigen( s ) , see also Transplantation
an tigens
fate in nlaCI’Ophdg.%, 290-304
SUBJECT INDEX
409
interaction with macroglobulins, 89-94
localization in lymphoid tissue, 288-
processing by vacuolar system, 285-
receptors, macroglobulins and, 105-
recognition, 214-217
synthetic, antibodies to, 4 1 4 3
290
304
106
mechanism of, 246-247
Antigenic competition, antibody synthe-
sis and, 276
Antigenic determinants, multiple, anti-
body synthesis and, 276-277
Antisera, bacterial carbohydrates, isola-
tion of, 30-37
B
Bacterial carbohydrates, antibodies, isola-
Bone marrow, transplantation, applica-
Bone marrow cells
tion from antisera, 30-37
tion, 255-257
differentiation of, 212-213
immunocompetent, 207-213
transfer to iinmunoincompetent recipi-
ents, 204-205
C
Carbohydrates, streptococcal, rabbit anti-
bodies to. 12-23
Cells,
see
also Immunocompetent cells,
Immunoincompetent cells
adherent, 273-275
antibody-forming, 228-232
distinction from antigen-reactive,
232-234
an tigen-reactive, 222-228
distinction from antibody-forming,
delayed hypersensitivity reaction and,
graft-versus-host reaction and, 247-
232-234
249-251
249
humoral immune response and, 213-
237
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410
SUBJECT
NDEX
immunological tolerance and, 251-255
interaction in induction of immune
response
cell-mediated,
242-243
humoral, 237-242
postulated mechanism,
243-244
macroglobulin interaction with, 97
transplantation rejection reaction and,
247-249
Compatibility assays, transplantation
Complement, macroglobulins and, 94-97
antigens, 157-173
D
Detergents, transplantation antigens and,
120-124
G
Genetics, macroglobulins and, 98-100
yA Globulins,
see
also Antibodies, Im-
munoglobulins, Macroglobulins
molecular size,
338-340
molecular size,
334337
overall shape and flexibility, 341-351
molecular size, 337-338
overall shape and flexibility, 351-356
Graft-versus-host reaction, cells involved,
yG Globulins
yM Globulins
247-249
H
Hemolytic focus assay, immune response
Hemolytic plaque assay, immune re-
Hypersensitivity
and,
205-206
sponse and, 205
delayed- ype
cells involved, 249-251
transplantation antigens and,
173-
181
I
Immune injury, lysosomes and,
306-321
Immune response
afferent limb, macrophages and, 285-
287
antibody-forming cell, 228-232
distinction from antigen-reactive
antigen-reactive cell and,
222-228
cell, 232-234
distinction from antibody-forming
cell,
232-234
cell-mediated
cell interactions,
242-243
cells involved, 246-251
hemolytic focus assay, 205-206
hemolytic plaque assay and, 205
humoral
cell interactions, 237-242
cells involved, 213-237
induction, cell interactions,
237-244
macroglobulins and, 104-106
other i n
vitro
assays,
206-207
radiation effects on, 244-246
detection techniques, 203-207
organ
of
origin, 207-212
two universes of, 272-273
Immunocompetent cells
Immunoglobulin( s) , see also Antibodies,
Globulins, Macroglobulins
allotypes,
6
amino acid sequence, 8-10
charge heterogeneity,
4-5
classes, subclasses and fight-chain
conformation, 341-366
recovery after dissociation, 367-375
half-molecules, reversible dissociation,
heavy and light chains
types, 3 4
370
dissociation and reversal,
371-375
properties
of, 363-366
heterogeneity, limited, 3-10
individual antigenic specificity of, 7-8
internal folding of, 356-362
molecular size, 334-341
overall shape and flexibility, 341-356
subunits
conformation of, 341-366
molecular size,
334341
Immunoincompetent recipients
bone marrow cell transfer
to,
204-205
lymphoid cell transfer to, 203-204
Immunological tolerance, cells affected,
251-255
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SUBJECT
INDEX
411
L
Lymphoid cells,
migration
in
vivo,
234-237
transfer to normal or immunoincompe-
Lymphoid tissue, antigen localization in,
Lysosomes
tent recipients, 203-204
288-290
immune injury
type I reaction, 306-309
type 11 reaction, 309-311
type
111
reaction, 311-316
type
IV
reaction, 316-321
iiiediation of tissue injury and, 304-
306
M
Macroglobulin( s
),
see also Antibodies,
Globulins,
Im~nunoglobulins
allelic markers and, 99
as antigen receptors, 105-106
biosynthesis of, 100-101
chemical properties, 71-73
complement system and, 94-97
control of antibody synthesis and, 106
distribution of, 101
functional properties of, 89-97
jdiotypic markers and, 99-100
immune response and, 104-106
interaction
antigens, 89-94
cells, 97
isolation methods
Macrophages
afferent limb
of
immune response and,
fate of antigen in, 290-304
humoral immune response and, 217-
Myeloma proteins, antibody activity, 43-
Myoglobin, antibodies to, 4 0 4 1
285-287
222
47
P
Paraproteins, antibody activity, 43-47
Pneumococcal capsular polysaccharides,
rabbit antibodies to, 29-30
Polypeptide chains, macroglobulin, 76-
85
Protein(
s )
macroglobulin interaction with, 97
macroglobulin-like, nonmammalian
species and, 102-104
Proteolysis
macroglobulins, 85-89
transplantatjon antigens and, 125-135
R
Rabbit
antibodies
pneumococcal capsular polysaccha-
streptococcal carbohydrates, 12-28
246
munoglobulins and, 366-370
rides, 29-30
Radiation, immune response and, 2 4 4
Random-coil formation, reversible, im-